Canine immunotherapeutics and uses thereof in cancer treatment

Abstract

Provided herein include new antibodies reactive for canine CD3, which are shown to substantially increase activation of canine T cells; new multi-specific immune cell engager molecules based on the canine CD3 antibodies, which are shown to effectively bind canine cancer antigens and recruit T cells for killing of the cancer cells; as well as new recombinant proteins derived from canine cartilage protein fragments and hydrogels thereof. Uses of these compositions are also provided, which include therapeutic uses of antibodies and multi-specific immune cell engager molecules for treatment of cancers or other diseases in dogs, as well as uses of hydrogels for local immunotherapy delivery for reduction of post-surgical recurrence risk, reduction of cancer at surgically inoperable sites, provision of palliation, and / or substitution for surgery.

Description

CANINE IMMUNOTHERAPEUTICS AND USES THEREOF IN CANCER TREATMENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63 / 691,151, filed September 5, 2024, the entirety of which is hereby incorporated by reference. REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing submitted as a computer readable form named “067505_000018WOPT_SequenceListing”, having a size in bytes of 163,367 bytes, and created on September 5, 2025. The information contained in this computer readable form is hereby incorporated by reference in its entirety. FIELD OF DISCLOSURE

[0003] This disclosure relates to modified canine CD3 antibodies with T-cell activation capabilities, multispecific immune cell engager molecules thereof, and coiled-coil protein hydrogel based on canine cartilage protein sequences. BACKGROUND

[0004] CD3 is engaged in the surface expression of the T-cell antigen receptor (TCR) and the signal transduction pathway resulting from MHC ligand binding to the TCR. When antigen presenting cells (APCs) activate T-cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane by the CD3 chains. CD3 is made up of a number of invariant subchains of the immunoglobulin superfamily. There are four subtypes of CD3, including CD3δ (delta), CD3ε (epsilon), CD3γ (gamma), and CD3ζ (zeta). CD3δ / CD3ε and CD3γ / CD3ε can form the TCR-CD3 complexes through the α / β chain of TCR as heterodimers. All CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domain. Upon TCR engagement, these motifs become phosphorylated by Src family protein tyrosine kinases LCK and FYN, resulting in the activation of downstream signaling pathways. In addition of this role of signal transduction in T-cell activation, CD3 chains may play additional roles.

[0005] Antibodies against CD3 molecules are thought to stimulate or block T cell activation signal transduction. These antibodies are believed to further eliminate effector T cells or induce a regulatory phenotype in T cells. Existing CA17.2A12 is a mouse anti-dog CD3 antibody, originally generated by immunizing mice with the T cell receptor complex 4901-5416-2522.3 Page 1 of 75067505-000018WOPT(TCR / CD3) isolated from canine thymocytes. Multi-specific (including bi-specific) antibody constructs conferring CD3 specificity for canines are also limited.

[0006] It is an object of the present disclosure to provide polypeptides with specificity against canine CD3 which are also capable of effectively binding and activating canine T cells.

[0007] It is another object of the present disclosure to provide antibody constructs with multi-specificity including bi-specificity against canine CD3 and disease antigens.

[0008] It is another object of the present disclosure to provide a composition for direct injection as a therapeutic composition into tumor parenchyma (for example skipping surgery), resection cavity, or peritumoral tissue of canine subjects with reduced immunogenicity, wherein the composition may also deliver therapeutic and / or prophylactic agents.

[0009] It is another object of the present disclosure to provide polypeptides with specificity against canine CD3 configured for treatment of auto-immune disease.

[0010] It is another object of the present disclosure to provide methods of treating subjects, especially canine subjects, with tumors, especially local cancers or solid tumors.

[0011] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed disclosure, or that any publication specifically or implicitly referenced is prior art. SUMMARY OF THE INVENTION

[0012] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0013] Various embodiments provide multispecific antibody constructs, wherein a multispecific antibody construct includes one or more of a first polypeptide comprising a canine CD3-binding molecule, and one or more of a second polypeptide capable of binding an antigen associated with a disease, wherein the canine CD3-binding molecule comprises complementarity-determining regions (CDRs) of a variable light (VL) domain and / or a variable heavy (VH) domain of an anti-dog CD3 antibody of clone CA17.8D4, CA17.6F9, or CA17.2A12 or an antibody having at least 95% sequence identity to the clone CA17.8D4, CA17.6F9, or CA17.2A12. In some embodiments, when the antigen associated with a disease comprises B7-H3, the canine CD3-binding molecule comprises the CDRs of the clone CA17.8D4, CA17.6F9, or both or does not comprise the CDRs of the clone CA17.2A12.4901-5416-2522.3Page 2 of 75 067505-000018WOPT

[0014] In some embodiments, the first polypeptide of the multispecific antibody construct comprises a Fab from one of the clones or an scFv or VHH derived from one of the clones.

[0015] In some embodiments, the second polypeptide of the multispecific antibody construct comprises a Fab from an antibody specific for a canine antigen or a VHH or scFv derived from the antibody. Exemplary canine antigens include but are not limited to PD-L1, the B7-H3, EphA2, CEACAM5, ROR1, HER2 / neu, CD20, PSMA, IGF-1R, EGFR, A33, Ep- CAM, or a combination thereof.

[0016] In some embodiments, the one or more first polypeptides of a multispecific antibody construct is linked to the one or more second polypeptides via a GlySer linker. In some embodiments, the first polypeptide and the second polypeptide are connected via a knob- in-hole heterodimeric IgG1 Fc.

[0017] Various embodiments provide methods of killing canine cancer cells or cancer cells in a canine subject in need thereof, and the methods include treating the canine cancer cells with, or administering to the canine subject, a multispecific antibody construct disclosed herein. In some embodiments, the methods further include treating the canine cancer cells with interferon-gamma, or further administering interferon-gamma to the canine subject. In some embodiments, the cancer cells comprise glioblastoma cells, melanoma cells, or soft-tissue sarcoma cells.

[0018] Various embodiments provide canine CD3-binding molecules, which include: a CD3-specific VL domain and / or a CD3-specific VH domain of one anti-dog CD3 antibody selected from clone CD17.2A12, CA17.6F9, CA17.8D4, or a variant of having at least 95% sequence identity to the clone CD17.2A12, CA17.6F9, CA17.8D4; and a constant domain (CL) of a human Ig kappa light chain connected to the CD3-specific VL domain, and / or three constant domains (CH1, CH2, CH3) and a hinge region of a human IgG1 heavy chain connected to the CD3-specific VH domain. In some embodiments, a variant of the CD17.6F9 comprises an amino acid substitution I1D at the VL domain according to the IMGT numbering system, that is at the N-terminus of the CD3-specific VL domain replacing Ile (I) with Asp (D);

[0019] In some embodiments, the CL of the human Ig kappa light chain has an amino acid sequence of SEQ ID NO:26; and / or the three constant domains and the hinge region of the human IgG1 heavy chain has an amino acid sequence of SEQ ID NO:25.

[0020] In some embodiments, the canine CD3-binding molecule is capable of immunospecifically binding to and activating canine T cells, characterized by expression of activation marker CD25 in the canine T cells.4901-5416-2522.3Page 3 of 75 067505-000018WOPT

[0021] Vector encoding a canine CD3-binding molecule disclosed herein are also provided, wherein the vector further encodes a first secretory signal sequence at the N-terminus relative to the CD3-specific VL domain and / or a second secretory signal sequence at the N- terminus relative to the CD3-specific VH domain. In some embodiments, a vector encodes a polypeptide comprising an amino acid sequence of one of SEQ ID NOs:38-44.

[0022] Cell expression systems are also provided, which include a cell engineered with a vector disclosed herein. In some embodiments, the cell is a hybridoma or hybridoma cell.

[0023] Methods are provided for inducing canine T cell activation, which include treating canine T cells or canine peripheral blood mononuclear cells (PBMCs) with a canine CD3-binding molecule disclosed herein. Preferably at least a portion of the canine PBMCs comprise T cells. In some embodiments, a method for inducing canine T cell activation further includes measuring an increased expression of CD25 in the canine T cells or canine PBMCs in response to the treatment with the canine CD3-binding molecule.

[0024] Compositions for use in canine subjects are also provided, which include (1) a multispecific antibody construct comprising one or more of a first polypeptide capable of binding canine CD3 and one or more of a second polypeptide capable of binding another antigen than CD3, and a polymeric matrix; or (2) the multispecific antibody construct, a cytokine such as interferon, and the polymeric matrix; or (3) a CD3-binding molecule and the polymeric matrix. Preferably, the compositions allow for controlled release of the active agent (e.g., multispecific antibody construct, cytokine, or CD3-binding molecule), such as slow release of the active agent over 1-3 weeks or 3-6 weeks or 6-9 weeks or longer. In various embodiments, the polymeric matrix is injectable. In some embodiments, the polymeric matrix comprises natural or synthetic polymers and is in the form of a hydrogel upon reconstitution in a medium. In some embodiments, the polymeric matrix comprises microparticles of polymers, which are suitable for injection and controlled release of active agent.

[0025] In some embodiments, the cytokine and the multispecific antibody construct or the CD3-binding molecule are differently associated with the polymeric matrix formed from the recombinant protein, such that they are released from the polymeric matrix at a different rate. In some embodiments, the associations differ by the degradable linkers used in linkage to the polymeric matrix. In some embodiments, the associations differ by the location of linkage, e.g., near surface or within core of the polymeric matrix.

[0026] These compositions can also be used in a method of killing cancer cells in a canine subject. They may also be used for treating cancer or an overgrowth disease in a canine subject in a method by injecting the compositions to tumor parenchyma of the subject.4901-5416-2522.3Page 4 of 75 067505-000018WOPT

[0027] Systems are also provided, which include: a polymeric matrix in a lyophilized form; one or more of a first polypeptide capable of binding canine CD3 and one or more of a second polypeptide capable of binding an antigen, preferably a cancer antigen, optionally the one or more first polypeptides and the one or more second polypeptides being associated as one molecule; a cytokine comprising interferon gamma and / or interferon alpha; a biocompatible medium; and one or more containers.

[0028] Additional embodiments provide new CD3-binding molecules, which comprise an antigen-binding fragment of an antibody, wherein the antigen-binding fragment comprises a CD3-specific variable light (VL) domain and / or a CD3-specific variable heavy (VH) domain, wherein: the CD3-specific VL domain comprises three complementarity determining regions (CDRs) having an amino acid sequence of SEQ ID NO:102, YAS, and SEQ ID NO:104, respectively, and the CD3-specific VH domain comprises three CDRs having an amino acid sequence of SEQ ID NOs:99, 100, and 101, respectively, or the CD3-specific VL domain and the CD3-specific VH domain comprise the CDRs of clone CA17.5F11. In further aspect, the CD3-specific VL domain comprises the amino acid sequence of SEQ ID NO:80, and the CD3- specific VH domain comprises the amino acid sequence of SEQ ID NO:79.

[0029] In another embodiments, the CD3-specific VL domain comprises three CDRs having an amino acid sequence of SEQ ID NO:84, GAS, and SEQ ID NO:86, respectively, and the CD3-specific VH domain comprises three CDRs having an amino acid sequence of SEQ ID NOs:81-83, respectively, or the CD3-specific VL domain and the CD3-specific VH domain comprise the CDRs of clone CA17.3G9. In further aspects, the CD3-specific VL domain comprises the amino acid sequence of SEQ ID NO:74, and the CD3-specific VH domain comprises the amino acid sequence of SEQ ID NO:73.

[0030] In yet another embodiments, the CD3-specific VL domain comprises three CDRs having an amino acid sequence of SEQ ID NO:90, GAS, and SEQ ID NO:92, respectively, and the CD3-specific VH domain comprises three CDRs having an amino acid sequence of SEQ ID NOs:87, 88, and 89, respectively, or the CD3-specific VL domain and the CD3-specific VH domain comprise the CDRs of clone CA17.9C5 or clone CA17.6B3. In further aspects, the CD3-specific VL domain comprises the amino acid sequence of SEQ ID NO:76, and the CD3-specific VH domain comprises the amino acid sequence of SEQ ID NO:75; respectively.

[0031] In some embodiments, the CD3-binding molecules are humanized. In some embodiments, the CD3-binding molecules comprise a human Ig kappa light chain polypeptide and / or a human IgG1 type heavy chain polypeptide.4901-5416-2522.3Page 5 of 75 067505-000018WOPT

[0032] These new CD3-binding molecules can also be used for inducing canine T cell activation.

[0033] These new CD3-binding molecules may also be provided with another polypeptide to form a multispecific antibody construct, wherein the other polypeptide is capable of immunospecifically binding an antigen associated with a disease.

[0034] Exemplary antigens can be expressed on tumor cells, wherein the tumor cell may be from a cancer selected from the group consisting of: breast cancer, prostate cancer, gastric cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma, and leukemia.

[0035] In other embodiments, the second polypeptide is capable of immunospecifically binding to a cell surface antigen, receptor or receptor ligand, wherein the cell surface antigen, receptor or receptor ligand comprises PD-L1, B7-H3, EphA2, CEACAM5, ROR1, HER2 / neu, CD20, PSMA, IGF-1R, EGFR, A33, Ep-CAM, or a combination thereof.

[0036] In yet another embodiment, the second polypeptide is capable of immunospecifically binding to a cell surface antigen, receptor or receptor ligand such as CD28, CD40, CD19, CD20, CD22, CD23, CD27, CD32B, CD38, CD79a, CD79b, CD80, CD86, LFA-I, LFA-3, and CFA-I, or a combination thereof.

[0037] In some embodiments, the multispecific antibody construct is in the form of connect scFv, wherein the first polypeptide comprises the CD3-binding molecule is in a form of single-chain Fv (scFv), connected to the second polypeptide that is in the form of scFv. In other embodiments, the first polypeptide and the second polypeptide are connected via a knob- in-hole heterodimeric IgG1 Fc, wherein the first polypeptide comprises the CD3-specific VL domain, the CD3-specific VH domain, and a first of a IgG1 type heavy chain, and wherein the second polypeptide comprises a VL domain specific for a cancer antigen, a VH domain specific for the cancer antigen, and a second of the IgG1 type heavy chain. In some embodiments, the first polypeptide and / or the second polypeptide comprises a Fc-silenced, canine IgG1 type heavy chain.

[0038] The multispecific antibody construct based on new CD3-binding molecules can also be used in a method of killing canine cancer cells or cancer cells in a canine subject, optionally in combination with a controlled-release matrix, such as being encapsulated in or covalently linked to the matrix.4901-5416-2522.3Page 6 of 75 067505-000018WOPT

[0039] Systems are provided including two or more of: a polymeric matrix in a lyophilized form; any multispecific antibody constructs disclosed herein; a cytokine comprising interferon gamma and / or interferon alpha; a biocompatible medium; and one or more containers. Optionally the system includes these components and a syringe or an injection device.

[0040] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. BRIEF DESCRIPTION OF THE FIGURES

[0041] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0042] Figures 1A-1G depict some exemplary CD3 antibodies reactive for canine CD3. Figure 1A is a chart summarizing sequences in figures 1B-1H. Figures 1B-1D depict antibodies whose variable regions are from clone CA17.2A12 (figure 1B), CA17.8D4 (figure 1C), or CA17.6F9 or variant thereof (figure 1D), with a mouse IgG1 chain or a mouse IgK chain. ) Figures 1E-1G depict antibodies whose variable domains are from clone CA17.2A12 (figure 1E), CA17.8D4 (figure 1F), or CA17.6F9 or variant thereof (figure 1G), with a human IgG1 chain and a human IgK chain.

[0043] Figure 2 depicts antibodies reactive for canine CD3 activated canine T-cells. In panel A, staining results with canine PBMCs by a canine CD3 (CA17.2A12) antibody of BioRad #MCA1774 (which comprises rat IgG), denoted as ‘2A12 WT’, wherein no increase in CD25+ cells compared to the no-antibody control shows that this commercially available antibody didn’t activate T-cells. In panel B, to test whether our canine CD3 antibodies activate T-cells (in terms of CD25+) or not, we treated canine PBMCs with our canine CD3 antibodies and stained against CD25. This graph shows that our antibody activates T-cells in canine PBMCs, despite this canine PBMCs had only 20% of T-cells to begin with.

[0044] Figure 3 depicts that our canine CD3 antibodies having human IgG1 constant domains bind canine T-cells. To test the binding of our CD3 antibodies on canine T-cells, we stained purified canine T-cells with our canine CD3 antibodies at different concentrations. This figure shows that our canine CD3 antibodies bind canine T-cells and affinity varies between antibodies and at different concentrations. ‘Control Ab’ is human IgG1 isotype control; whereas the other three groups in this figure have human IgG1 type antibodies.4901-5416-2522.3Page 7 of 75 067505-000018WOPT

[0045] Figures 4A-4L depict amino acid sequences of polypeptides expressed by expression constructs encoding bispecific T cell engager (BiTE or BTE) molecules comprising a binding domain for immune cells such as T-cells (e.g., an anti-canine CD3 scFv selected from clones CA17.2A12, CA17.6F9 or variant of CA17.6F9, or CA17.8D4) and a binding domain for cancer (e.g., an anti-PDL1 scFv such as that from clone 4F9, clone 5F12, or atezolizumab, or an anti-B7H3 such as that from enoblituzumab also known as MGA271), wherein the two binding domains are linked via a GlySer linker. Figure 4A depicts BiTE_cfPDL1_4F9_scFv_VLVH-CD3_CA17.2A12_scFv_VHVL-His. Figure 4B depicts BiTE_cfPDL1_4F9_scFv_VLVH-CD3_CA17.6F9_scFv_VHVL-His. Figure 4C depicts BiTE_cfPDL1_4F9_scFv_VLVH-CD3_CA17.8D4_scFv_VHVL-His. Figure 4D depicts BiTE_cfPDL1_5F12_scFv_VLVH-cfCD3_CA17.2A12_scFv_VHVL-His. Figure 4E depicts BiTE_cfPDL1_5F12_scFv_VLVH-cfCD3_CA17.6F9_scFv_VHVL-His. Figure 4F depicts BiTE_cfPDL1_5F12_scFv_VLVH-cfCD3_CA17.8D4_scFv_VHVL-His. Figure 4G BiTE_cfPDL1_Atezo_scFv_VLVH-cfCD3_CA17.2A12_scFv_VHVL-His. Figure 4H depicts BiTE_cfPDL1_Atezo_scFv_VLVH-cfCD3_CA17.6F9_scFv_VHVL-His. Figure 4I depicts BiTE_cfPDL1_Atezo_scFv_VLVH-cfCD3_CA17.8D4_scFv_VHVL-His. Figure 4J depicts BiTE_cfB7H3_MGA271_scFv_VLVH-cfCD3_CA17.2A12_scFv_VHVL-His. Figure 4K depicts BiTE_cfB7H3_MGA271_scFv_VLVH-cfCD3_CA17.6F9_scFv_VHVL- His. Figure 4L depicts BiTE_cfB7H3_MGA271_scFv_VLVH- cfCD3_CA17.8D4_scFv_VHVL-His.

[0046] Figures 5A and 5B depict structure (5A) and yield of production (5B) of canine bi-specific T-cell engager (BTE) molecules. In figure 5A, canonical format of BTEs: ScFv of cancer antigen binding domain (e.g., anti-PDL1, anti-B7H3) is connected to ScFv of immune cell binding domain (e.g., anti-CD3) via a GlySer linker. Figure 5B is a heatmap of BTE production.

[0047] Figures 5C-5E depict that canine BTEs kill canine glioblastoma cells (G06A line is glioblastoma cell line). Figure 5C shows T-cell killing assay with BTE 2.5 ug / ml using canine activated T-cells and brain tumors. Figure 5D shows T-cell killing assay with MDT- 002422 at different concentrations with canine IFN-gamma 100ng / ml using canine activated T-cells and brain tumors. Figure 5E shows T-cell killing assay with MDT-0024220.1ug / ml with canine IFN-gamma 100ng / ml using canine activated T-cells and brain tumors.

[0048] Figure 5F depict that canine BTEs kill canine glioma cell line SDT3G.

[0049] Figures 6A-6B depict that canine BTEs killed canine melanoma cells. Figure 6A: T-cell killing assay with MDT-002422 at different concentrations using canine activated4901-5416-2522.3Page 8 of 75 067505-000018WOPTT-cells and melanoma cells. Figure 6B: T-cell killing assay with MDT-0024220.02ug / ml with canine IFN-gamma 100ng / ml using canine activated T-cells and melanoma cells.

[0050] Figures 7A and 7B show amino acid sequences encoded by vectors expressing coiled-coil recombinant proteins having canine cartilage protein-derived fragments (MDT002397 having the C-terminus Csy-Asp-Ala motif replaced with Ser-Asp-Ala (figure 7A); MDT002398 having the C-terminus Csy-Asp-Ala motif removed (figure 7B)).

[0051] Figure 7C depicts the hydrogels formed from the coiled-coil recombinant proteins based on canine cartilage protein fragments retain a fluorescent protein mScarlet (as a mimic of protein-based therapy), wherein hydrogel was formed by reconstituting the lyophilized recombinant protein with mScarlet-containing phosphate buffer solution (PBS). Then, bovine serum albumin (BSA)-containing PBS was added on top of the hydrogel to confirm the hydrogel retains mScarlet.

[0052] Figure 7D shows release kinetics of 5% hydrogels. We embedded BTE into the hydrogel, added BSA-containing PBS, harvested this BSA-containing PBS at different time points, and ran flowcytometry-based sandwich ELISA to measure quantity of BTE released from the hydrogel.

[0053] Figure 7E depicts the release of canine IFN-g (cIFN-g) from the hydrogel. We embedded 1ug cIFN-g into 100ul of the hydrogel and detected cIFN-g release into the supernatant by cIFN-g ELISA.

[0054] Figure 8 is a graphical abstract of Hydrogel-loaded with ImmunoTherapy (HIT). HIT is injected into tumor tissue or peri-tumoral tissue. The hydrogel releases IFNg for the first few days to change tumor microenvironment to immunosuppressive to immunoactive and PD-L1:CD3 BTE for a couple of weeks to eliminate the residual cancer cells.

[0055] Figure 9 depicts IFNg and PD-L1:CD3 BTE release from rat cartilage-based hydrogel and tunability of release. Panel A) Image of coiled-coil hydrogel, Panel B) Lyophilized form of our hydrogel, Panel C) Photo of the hydrogel exiting the needle of Insulin syringe. Panels D & E) Both IFNg 100ng and PD-L1:CD3 MaxiBTE were loaded into 100ul of the mouse hydrogel. Panel D) IFNg release into the supernatant was detected by IFNg ELISA. 100% release is defined by the plateau. Panel E) PD-L1:CD3 BTE release into the supernatant was detected by CD3 / PDL1 sandwich ELISA. Panel F) Protein release from the hydrogel is tunable by changing the weight % of the hydrogel. 5%, 10%, and 12.5% wt hydrogel (100ul) were formed in 1.5ml Eppendorf tubes and 900ul 0.2% BSA-containing PBS was loaded on top of the hydrogel. Less degradation was seen at day 14 in higher % gels. Panel G) PD-L1:CD3 BTE release into the supernatant was observed by CD3 / PD-L1 ELISA.4901-5416-2522.3Page 9 of 75 067505-000018WOPT

[0056] Figure 10 depicts a plan for canine clinical trial. On the left, injection methods are compared: Injecting hydrogel into tumor resection cavity or into tumor parenchyma (skipping surgery), or Injecting hydrogel “columns” into the tumor or peritumoral tissue. A proposed treatment procedure is also depicted.

[0057] Figures 11A and 11B are schematics of multispecific (more specifically, bispecific) antibody constructs including a CD3 binder (clone 17.2A12 in figure 11A, or clone 17.2A12, 17.6F9 or 17.8D4 in figure 11B) and a binder for tumor-associated antigen such as PD-L1, B7H3, or EphA2. A construct formed by connecting two scFv is coined as “canonical” bispecific T-cell engager (BTE). A constructed formed by a heterodimeric knob-in-hole IgG1 type Fc, wherein one monomer comprises a CD3-binding VH and CD3-binding VL kappa light chain, and the other monomer comprises an scFv against a tumor-associated antigen, is coined as “Maxi” BTEs.

[0058] Figure 12A depicts the effect of T-cell engagers (also referred to as multispecific antibody constructs) on activating T-cells, when canine activated T-cells (ATCs) were co-cultured with STSA-1 tumor (canine soft tissue sarcoma) cells at the ratio of 5:1 effector:tumor. BTEs (SCRI-001959 (“1959”), SCRI-001968 (“1968”), and SCRI-002118 (“2118”) whose construct was depicted in figure 11A) were titrated. CD25 levels were evaluated as a T-cell activation marker at 48 hours. PDL1:CD3 (“1959”) and B7H3:CD3 (“1968”) canonical TCEs activated T cells that were cultured with tumor cells in dose dependent manner, whereas Fc-containing PDL1:CD3 (“2118”) TCE did not.

[0059] Figure 12B depicts the effect of T-cell engagers (also referred to as multispecific antibody constructs) on activating T-cells, when canine PBMCs were co-cultured with STSA- 1 tumor (canine soft tissue sarcoma) cells at the ratio of 5:1 effector:tumor. BTEs (i.e., PDL1:CD3 (“1959”) and B7H3:CD3 (“1968”)) were titrated. CD25 levels in CD5+ cells as a T-cell activation marker were evaluated at 48 hours. Both PDL1:CD3 and B7H3:CD3 TCEs activated T cells that were cultured with tumor cells in dose dependent manner.

[0060] Figure 13A depicts the efficacy of TCE “1959” at various concentrations in T- cell killing (TCK) assays, with canine activated T-cells. This graph plots the normalized relative tumor cell counts over time course. This data shows that the TCE is effective at killing canine soft tissue sarcoma cells in a dose-dependent manner.

[0061] Figure 13B depicts the efficacy of TCE “SCRI-001961” (or “1961”) at various concentrations in T-cell killing (TCK) assays, with canine activated T-cells. This graph plots the normalized relative tumor cell counts over time course. This data shows that the TCE is effective at killing canine soft tissue sarcoma cells in a dose-dependent manner.4901-5416-2522.3Page 10 of 75 067505-000018WOPT

[0062] Figure 13C depicts the efficacy of TCE “SCRI-001965” (or “1965”) at various concentrations in T-cell killing (TCK) assays, with canine activated T-cells. This graph plots the normalized relative tumor cell counts over time course. This data shows that the TCE is effective at killing canine soft tissue sarcoma cells in a dose-dependent manner.

[0063] Figure 13D depicts the efficacy of TCE “1968” at various concentrations in T- cell killing (TCK) assays, with canine activated T-cells. This graph plots the normalized relative tumor cell counts over time course. This data shows that the TCE is effective at killing canine soft tissue sarcoma cells in a dose-dependent manner.

[0064] Figure 14 is a schematic of four exemplary multispecific antibody constructs, comprising a CD3-binding domain and: both of a B7H3-binding domain and a PD-L1-binding domain (“SCRI-002320” or “2320”), a ROR1 VHH (“SCRI-002324” or “2324”), or both a PD- L1-binding domain and two ROR1 VHH (“SCRI-002325” or “2325”, and “SCRI-002326” or “2326”).

[0065] Figures 15A and 15B depict the efficacy of TCEs (SCRI-002320 and SCRI- 002324 in figure 15A; SCRI-002325 and SCRI-002326 at figure 15B) at various concentrations in canine activated T-cell killing assays. The graphs plot the normalized relative tumor cell counts over time course. This data shows that the TCEs are effective at killing canine soft tissue sarcoma cells in a dose-dependent manner.

[0066] Figure 16A depicts the efficacy of TCEs (“1959,” “1961,” “1968,” and “2320”) in T-cell (activated canine T-cell) killing assays using 5:1 (upper panel) and 1:1 (lower panel) tumor:effector cell ratios. The graphs plot % tumor cell death at different TCE concentrations at Day 5. This data shows that the TCEs are effective at killing canine soft tissue sarcoma cells in a dose-dependent manner.

[0067] Figure 16B depicts the efficacy of TCEs (“1959,” “1968,” and “2118”) in T- cell (canine activated T-cell) killing assays. The graphs plot the normalized relative tumor cell counts over time course. This data shows that the TCEs are effective at killing canine soft tissue sarcoma cells in dose-dependent manner.

[0068] Figure 16C depicts the efficacy of TCEs (“1959,” “1968,” and “2118”) in T- cell (canine activated T-cell) killing assays using 5:1 and 1:1 tumor:effector cell ratios on canine glioblastoma (GMB) cells. The graphs plot % tumor cell death at different TCE concentrations at Day 5. This data shows that the TCEs are effective at killing canine soft tissue sarcoma and glioblastoma cells in a dose-dependent manner.

[0069] Figure 17 depicts a schematic of binding sites for various mAb clones CA15. / CA17. / CA20. on T cell receptor complex. This provides a visual map of the hybridomas4901-5416-2522.3Page 11 of 75 067505-000018WOPTfor finer specificity. CA15 series are all TRB specific; CA17 series are all CD3 specific; and CA20 series are all TRG / TRD specific.

[0070] Figure 18A depicts the effect of exemplary anti-canine CD3 antibodies with a human IgG1 Fc domain on activating T-cells in PBMCs. We cultured canine peripheral blood mononuclear cells (PBMCs) and titrated anti-canine CD3 Abs for 24 hours and measured CD25 levels as a T-cell activation marker. CA17.2A12 showed higher CD25 level compared to negative control Ab group. New CD3-binders (3G9, 9C5, 6B3, 5F11) also showed higher CD25 levels compared to the negative control Ab group. Notably, clone CA17.5F11 showed the highest CD25 level and as high as 2A12.

[0071] Figure 18B depicts a summary of the constant domains comprised of human IgG1 type heavy chain and human kappa type light chain for the various canine CD3-specific antibodies (clones CA17.3G9, CA17.9C5, CA17.6B3, and CA17.5F11).

[0072] Figures 18C, 18F, 18G, and 18J show the VH and VL sequences and the “string of pearl” representation using the IMGT numbering system of CA17.3G9 (figure 18C), CA17.9C5 (figure 18F), CA17.6B3 (figure 18G), and CA17.5F11 (figure 18J), respectively.

[0073] Figures 18D, 18E, 18H, and 18I show the encoded heavy chain and light chain polypeptides, separated by a colon (:), of the clones CA17.3G9 (figure 18D), CA17.9C5 (figure 18E), CA17.6B3 (figure 18H), and CA17.5F11 (figure 18I), respectively, which includes the signal peptide (highlighted at N-terminus) and CDR sequences (highlighted).

[0074] Figure 19A is a schematic depicting exemplary BTEs based on a new canine CD3-binding scFv and another scFv specific for PD-L1, B7H3, EphA2, or CEACAM5.

[0075] Figure 19B is a schematic depicting BTEs based on an scFv or Fab specific for CD28 or a CD40 ligand and an scFv (optionally linked to an Fc domain) or Fab specific for PD-L1.

[0076] Figures 20A-20M show the polypeptide sequences of various multispecific antibody constructs including signal peptide. Figure 20A depicts MaxibAb_cfCD28_VLVH- cfIgG1_Knob-His and MaxibAb_cfPDL1_Atezo-cfIgG1_Hole. Figure 20B depicts MaxibAb_cfCD28_VH-cfIgG1_Knob, MaxibAb_cfPDL1_Atezo-cfIgG1_Hole, and MaxibAb_cfCD28_VL-cfCH1. Figure 20C depicts MaxibAb_cfCD28_VLVH-cfIgG1_Knob- His, MaxibAb_cfPDL1_459_Merck_VH-cfIgG1_Hole, and MaxibAb_cfPDL1_4F9_Merck_VL-cfkappa. Figure 20D depicts MaxibAb_cfCD28_VH- cfIgG1_Knob, MaxibAb_cfPDL1_4F9_VLVH-cfIgG1_Hole, and MaxibAb_cfCD28_ VL- cfCH1. Figure 20E depicts MaxibAb_hsCD40L-cfIgG1_Knob-His and MaxibAb_cfPDL1_Atezo-cfIgG1_Hole. Figure 20F depicts4901-5416-2522.3Page 12 of 75 067505-000018WOPTBiTE_cfPDL1_459_Merck_VLVH-cfCD28_ScFV_Philogen-His. Figure 20G depicts BiTE_cfPDL1_Atezo_scFv_VLVH-cfCD28_ScFV_Philogen-His. Figure 20H depicts BiTE_cfPDL1_Atezo_scFv_VLVH-hsCD40L-His. Figure 20I depicts BiTE_cfPD- L1_4F9_ScFv_VLVH-cfCD3_17.5F11_ScFv_VH_VL_His. Figure 20J depicts BiTE_cfB7H3 MGA271 ScFv :cfCD3 17.5F11 ScFv-VH-VL_His. Figure 20K depicts BiTE_cfEphA2 208 VLVH ScFv:cfCD3 17.5F11 ScFv-VH-VL_His. Figure 20L depicts BiTE_humanCEACAM5_scFv_VLVH-cfCD3_CA17.5F11_scFv_VHVL-His. Figure 20M depicts BiTE_humanCEACAM5mini_immune_scFv_VLVH- cfCD3_CA17.5F11_scFv_VHVL-His. DESCRIPTION

[0077] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rded., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2nded. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511; Queen et al. U. S. Patent No.5,585,089; and Riechmann et al., Nature 332: 323 (1988); U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep;23(9):1126- 36).

[0078] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described. For purposes of the present disclosure, the following terms are defined below.

[0079] The term “CD3-binding molecule” denotes a molecule capable of immunospecific binding to CD3 through at least one antigen recognition site (e.g., an antigen- binding domain of an antibody) located in the variable region of the molecule. In various embodiments, a “CD3-binding molecule” disclosed herein is capable of immunospecific4901-5416-2522.3Page 13 of 75 067505-000018WOPTbinding to the CD3 of a dog, or canine CD3. In various embodiments, a CD3-binding antibody disclosed herein is immunospecific to canine CD3. The term “CD3-binding molecule” encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, “BiTEs®,” “DART™” diabody molecules and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.

[0080] The term “monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non- naturally occurring) that are involved in the selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The term “monoclonal antibody” encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2 Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity and the ability to bind to an antigen. It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody.”

[0081] The term “humanized antibody” refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either complete variable domains fused onto constant domains or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domains. Antigen binding sites may be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains. Another approach focuses not only on providing human-derived constant regions, but modifying the variable regions as well so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the antigens in4901-5416-2522.3Page 14 of 75 067505-000018WOPTquestion and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular antigen, the variable regions can be “reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified.

[0082] In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. As disclosed below, the preferred antibodies of the present invention have specific identified CDRs. The present invention, however, contemplates equivalent antibodies having altered CDRs.

[0083] The phrase “string of pearls” refers to the IMGT Colliers de Perles (Necklaces of Pearls), which are two-dimensional graphical representations of immunoglobulin (antibody) or T-cell receptor domains. In IMGT Collier de Perles, each bead represents an amino acid, and the shape indicates whether it belongs to a beta strand (circles) or another region (squares). The IMGT numbering provides standardized positions for comparing different sequences, regardless of the species or receptor type. A variable (V) domain is numbered from 1 to 128 and includes the following regions: (1) Framework Regions (FR-IMGT): These are the conserved structural regions of the domain. FR1-IMGT: Positions 1–26. FR2-IMGT: Positions 39–55. FR3-IMGT: Positions 66–104. FR4-IMGT: Positions 118–128; and (2) Complementarity-Determining Regions (CDR-IMGT): These are the hypervariable loops that are critical for antigen binding. They are delimited by the framework regions. CDR1- IMGT: Positions 27–38. CDR2-IMGT: Positions 56–65. CDR3-IMGT: Positions 105–117. Conserved amino acids, which are crucial for the overall protein fold, are always at the same positions. For example, the cysteine residues forming a disulfide bridge are consistently at positions 23 and 104. CDRs are anchored by specific amino acids in the framework regions, which are shown in squares. This makes their boundaries precise and consistent. The IMGT numbering uses the longest possible sequence as a reference and adds gaps (represented by hatched circles or squares) for shorter sequences to maintain the alignment. For CDR3s shorter than 13 amino acids, gaps are placed at the top of the loop. For sequences with insertions longer than the reference, the system adds sub-positions with decimals, such as 112.1, 112.2, etc.,4901-5416-2522.3Page 15 of 75 067505-000018WOPTtypically in the CDR3 loop. Arrows indicate the direction of the beta strands in the 3D structure.

[0084] An antibody or a polypeptide is said to “immunospecifically” or equivalently, “specifically” bind a region of another molecule (i.e., an epitope) if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with that epitope relative to alternative epitopes. For example, an antibody that specifically binds to a CD3 epitope is an antibody that binds this CD3 epitope with greater affinity, avidity, more readily, and / or with greater duration than it binds to other CD3 epitopes or non-CD3 epitopes.

[0085] The term “linker” or “linker moiety” refers broadly to a chemical structure that is capable of linking or joining together two polypeptide subunits or linking a polypeptide to another group. “Linker” in a polypeptide can be a short peptide, such as a dimer of two amino acids, a tri-mer of three amino acids, a 4-mer of four amino acids, a 5-mer of five amino acids or a peptide selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:152), and (GGGGXλ(SEQ ID NO:153))n wherein Xλis Q, A, E or S and n=1-5 or an integer larger than 5. In some embodiments, the amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO:4))n where n is an integer between 1 and 5, thereby an amino acid linker of 25 amino acids or shorter in length. Additional examples include (XλGGGG (SEQ ID NO:154))n wherein Xλ is Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5, (GXλGGG (SEQ ID NO:155))nwherein Xλis Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5, (GGXλGG (SEQ ID NO:156))n wherein Xλ is Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5, (GGGXλG (SEQ ID NO:157))n wherein Xλ is Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5. Still additional examples include (XλGGG)n wherein Xλis Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5, (GXλGG)n wherein Xλ is Q, A, E or S and n=1- 5 or in some embodiments, an integer larger than 5, (GGXλG)n wherein Xλis Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5, (GGGXλ)n wherein Xλ is Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5. Still additional examples include (XλGG)n wherein Xλ is Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5, (GXλG)n wherein Xλis Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5, (GGXλ)n wherein Xλ is Q, A, E or S and n=1-5 or in some embodiments. Still additional examples include (XλG)n wherein Xλis Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5, (GXλ)n wherein Xλ is Q, A, E or S and n=1-5 or in some embodiments, an integer larger than 5. As further examples, the short polypeptide is 1, 2, 3, or 4 amino acids, or flexible linkers or amino acid chain of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,4901-5416-2522.3Page 16 of 75 067505-000018WOPT25, 30, or 35 amino acids. The term “association,” with regard to an antibody, includes covalent and non-covalent attachment or binding of an agent (e.g., coiled-coil canine cartilage derived protein) to the antibody. The antibody can be associated with an agent (e.g., coiled-coil canine cartilage derived protein) by direct binding or indirect binding via attachment to a common platform.

[0086] The term “individual” refers to a vertebrate animal, preferably a mammal. Mammals include, but are not limited to, humans, farm animals, sport animals, pets, primates, mice and rats. In the most preferred embodiment, the term individual denotes a dog.

[0087] The terms “treatment” or “treating” denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing inflammation or an autoimmune response, reducing the proliferation of (or destroying) cancerous cells or other diseased cells, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of individuals.

[0088] “Flexible domain” refers to a peptidyl flexible linker. In various embodiments, the flexible domain is an “XTEN” linker, which is a class of highly expressed, water-soluble, and chemically stable, intrinsically disordered / unstructured polypeptide, which comprises a pseudorepeat of amino acids. It may be evolved to “eXTENd” the in vivo half-life of fused peptides / protein therapeutics, as a biological alternative to PEGylation. In some embodiments, XTEN is comprised of 36-residue pseudo-repeats of only six amino acids (A, E, G, P, S, and T), and it lacks hydrophobic residues typically associated with histocompatibility complex class II-driven immune responses, amino acids that typically bind cell membranes, and cysteines that can undergo disulfide crosslinking. In some embodiments, XTEN is comprised of a pseudorepeat of 5-7 different amino acids. In some embodiments, XTEN comprises a quantity of 36-1,728 amino acids. In some embodiments, XTEN comprises about 144 residues. In some embodiments, XTEN comprises a quantity of more than 1,728 amino acids. In some embodiments, XTEN comprises a quantity of fewer than 36 amino acids. In some embodiments, a short XTEN (e.g., shorter than 36 amino acids) does not permit annealing with a second coil on the same molecule, but permits annealing with a coil on another molecule. In some embodiments, an exemplary XTEN comprises an amino acid sequence of SEQ ID NO:62.4901-5416-2522.3Page 17 of 75 067505-000018WOPT

[0089] “Intrinsically unstructured” refers to a protein or peptide sequence that lacks ordered secondary or tertiary structure. For example, an intrinsically unstructured protein or peptide sequence lacks an alpha helix, a beta sheet, or a fixed or ordered tertiary structure. The intrinsically unstructured protein can adopt multiple configurations.

[0090] “Pseudorepeat” refers to a defined number of amino acids which are not the same, and which are repeated in an order which is not regular. For example, a pseudorepeat can be a repeat of 5, a repeat of 6, or a repeat of 7 different amino acids, or amino acids which have a different identity. In a pseudorepeat of amino acids, the total number of amino acids comprises different amino acids (e.g., 5, 6, or 7), which are repeated to make up the total number of amino acids, but wherein the different amino acids are repeated such that no intentional pattern of the repeated amino acids exists. For example, the repeat of a pseudorepeat of 5 different amino acids having the generic identity a, b, c, d, and e does not comprise a pattern such as . . . a-b-c-d-e-a-b-c-d-e-a-b-c-d-e.. . ; ... a-a-a-b- b-b-c-c-c-d-d-d-e-e-e. . . ; or any other regular type pattern.

[0091] In various embodiments, the different amino acids in a pseudorepeat are individually repeated to make up the total number of amino acids of the peptide or protein sequence. For example, a protein comprising 5 different amino acids which make up 100 total amino acids, can be made up of an equal amount of each of the 5 different amino acids, or can be made up of an amount of the 5 different amino acids which is not the same as between any or all of the 5 different amino acids. For example, a protein comprising 100 total amino acids can be comprised of 20 residues of amino acid “a,” 20 residues of amino acid “b,” 20 residues of amino acid “c,” 20 residues of amino acid “d,” and 20 residues of amino acid “e,” when the different amino acids exist in the protein at an identical or equal amount. Alternatively, the different amino acids can exist in the protein in an amount which is not identical or equal. For example, a protein comprising 100 total amino acids can consist of 20 residues of amino acid “a,” 15 residues of amino acid “b,” 35 residues of amino acid “c,” 10 residues of amino acid “d,” and 20 residues of amino acid “e”. The quantity of the most frequent amino acid can be up to about 5 times the quantity of the least frequent amino acid, up to about 4 times the quantity of the least frequent amino acid, up to about 3 times the quantity of the least frequent amino acid, or up to about 2 times the quantity of the least frequent amino acid.

[0092] In some embodiments, the flexible domain consists of a pseudorepeat of 6 different amino acids, wherein one amino acid has a negatively charged side-chain at physiological pH (e.g., aspartic acid or glutamic acid); two different amino acids have a polar4901-5416-2522.3Page 18 of 75 067505-000018WOPTside chain (e.g., serine, threonine); and three different amino acids have a nonpolar side chain (e.g., glycine, alanine, proline).

[0093] “Multispecific” refers to specificity to at least two molecules or at least two epitopes of one molecule. In various embodiments, multispecific molecules include bispecific molecules, which are immunospecific to two antigens or two epitopes of an antigen. The multispecific (or in some instances trispecific or bispecific) molecules of the present disclosure will be capable of binding the CD3 and also to a second (or additional) and different antigen(s) or epitope(s). The second antigen or epitope is preferably a tumor antigen expressed on a tumor cell. Such tumor cells may be from cancers, for example, breast cancer, prostate cancer, gastric cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma, or leukemia. The additional antigens or epitopes are preferably cell surface tumor antigens or epitopes (such as: 17-1A, A33, adult erythrocyte primary endoderm I antigen, alpha fetoprotein, an envelope antigen of an RNA tumor virus, bladder tumor oncofetal antigen, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6, Burkitt's lymphoma antigen-38.13, CA125, CD18, CD19, human B- lymphoma antigen-CD20, CD22, CD33, CD44, CD52, CEA, CO17-1A, CTA-1, CTLA-4, epidermal growth factor receptor, Ep-CAM, EphA2, receptor tyrosine kinase like orphan receptor 1 (ROR1), fetal erythrocyte I antigen, fibrosarcoma antigen, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, GICA 19-9, gp IIIb / IIIa, gp72, HER1, HER-2 / neu, HER3, HER4, high molecular weight melanoma antigen, HLA-DR antigen, human leukemia T cell antigen-Gp37, human lung carcinoma antigen L20, human lung carcinoma antigen L6, human milk fat globule antigen, IgE, KS 1 / 4 pan-carcinoma antigen, LEA, lung adenocarcinoma F3 antigen, malignant human lymphocyte antigen-APO-1, melanoma antigen gp75, melanoma-associated antigen p9′7, neoglycoprotein, nuC242, polymorphic epithelial mucin antigen, prostate specific antigen, prostate specific membrane antigen, prostatic acid phosphate, SK-1 antigen, TAG-72, T-antigen, tumor antigen CA125, tumor antigen MUC1, tumor-specific transplantation type of cell-surface antigen, vascular endothelial growth factor, vascular endothelial growth factor-receptor, and αvβ3). Alternatively, such additional antigens or epitopes may be associated with a pathogen (such as: hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus,4901-5416-2522.3Page 19 of 75 067505-000018WOPTechinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, small pox, Epstein Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), viral miningitis, viral encephalitis, dengue, small pox; mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi, Bacillus anthraces, Streptococcus, Staphylococcus, Mycobacterium, tetanus, pertissus, cholera, plague, diptheria, chlamydia, and legionella; leishmania, kokzidioa, trypanosoma or malaria; chlamydia and rickettsia. In some embodiments, multispecific molecules may be capable of immunospecifically binding to three or more antigens or three or more epitopes. The term bi-specific T-cell engager, also referred to as bi- specific antibody construct, refers to a single polypeptide chain molecule that having two antigen-binding domains, one of which binds to a T-cell antigen and the second of which binds to an antigen present on the surface of a target.

[0094] Methods of making monoclonal antibodies are known in the art. One method which may be employed is the method of Kohler, G. et al. (1975) “Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity,” Nature 256:495-497 or a modification thereof. Typically, monoclonal antibodies are developed in non-human species, such as mice. In general, a mouse or rat is used for immunization but other animals may also be used. The antibodies are produced by immunizing mice with an immunogenic amount of cells, cell extracts, or protein preparations that contain human CD3. The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancerous cells, nucleic acids, or tissue.

[0095] In one embodiment, monoclonal antibodies that bind to CD3 are obtained by using host cells that over-express CD3 as an immunogen. Such cells include, by way of example and not by limitation, human T cells.

[0096] To monitor the antibody response, a small biological sample (e.g., blood) may be obtained from the animal and tested for antibody titer against the immunogen. The spleen and / or several large lymph nodes can be removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or to a well coated with the antigen. B-cells, expressing membrane- bound immunoglobulin specific for the antigen, will bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, can then be fused with myeloma cells (e.g., X63-Ag8.653 and those from the SaIk Institute, Cell Distribution Center, San Diego, Calif.). Polyethylene glycol (PEG) may be used to fuse spleen or lymphocytes with myeloma cells to form a hybridoma. The hybridoma is then cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, otherwise known as4901-5416-2522.3Page 20 of 75 067505-000018WOPT“HAT medium”). The resulting hybridomas are then plated by limiting dilution, and are assayed for the production of antibodies that bind specifically to the immunogen, using, for example, FACS (fluorescence activated cell sorting) or immunohistochemistry (IHC) screening. The selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in mice).

[0097] As another alternative to the cell fusion technique, Epstein-Barr Virus (EBV)- immortalized B cells may be used to produce monoclonal antibodies of the subject invention. The hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti- immunogen activity by conventional assay procedures (e.g., FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, etc.).

[0098] In another alternative, anti-CD3 monoclonal antibody and any other equivalent antibodies can be sequenced and produced recombinantly by any means known in the art (e.g., humanization, use of transgenic mice to produce fully human antibodies, phage display technology, etc.). In one embodiment, anti-CD3 monoclonal antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use.

[0099] The polynucleotide sequence of anti-CD3 monoclonal antibody and any other equivalent antibodies may be used for genetic manipulation to generate a “humanized” antibody, to improve the affinity, or other characteristics of the antibody.

[0100] The disclosure includes variations to anti-CD3 antibodies and their binding fragments. Examples of modified polypeptides (or variants of polypeptides) include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs. Amino acid residues which can be conservatively substituted for one another include but are not limited to: glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine; lysine / arginine; and phenylalanine / tryosine. These polypeptides also include glycosylated and nonglycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Preferably, the amino acid substitutions would be conservative, i.e., the substituted amino acid would possess similar chemical properties as that of the original amino acid.

[0101] cDNAs encoding anti-CD3 antibodies or other CD3-binding molecules can be obtained by reverse transcribing the mRNAs from a particular cell type according to standard4901-5416-2522.3Page 21 of 75 067505-000018WOPTmethods in the art. The synthesized cDNAs may then be introduced into an expression vector to produce the antibody or protein of interest in cells of a second type. It is implied that an expression vector must be replicable in the host cells either as an episome or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and cosmids.

[0102] The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.

[0103] Any host cells capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include but are not limited to COS, HeLa, and CHO cells. Preferably, the host cells express the cDNAs at a level of about 5-fold higher, more preferably 10-fold higher, even more preferably 20-fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells. Screening the host cells for a specific binding to CD3 is effected by an immunoassay or FACS. A cell over-expressing the antibody or protein of interest can be identified.

[0104] Various embodiments provide a composition or combination comprising one, two, or more types of canine-specific immunomodulating agents and a polymeric matrix for controlled release or another pharmaceutically acceptable carrier, wherein the canine-specific immunomodulating agents comprises (1) one or more multispecific antibody constructs, at least one of which comprises a canine CD3-specific binding domain, (2) a canine CD3-specific binding polypeptide, or a combination of (1) or (2) and (3) a cytokine; and wherein the matrix for controlled release comprises hydrogel or another slow-release formulation. In preferable embodiments, the composition or the matrix is injectable. In some embodiments, the matrix is a hydrogel or comprises hydrogel-forming polymer. In some embodiments, the matrix comprises a shear-thinning hydrogel. In some embodiments, the matrix comprises a coiled-coil recombinant protein hydrogel. In further embodiments, the matrix comprises a coiled-coil recombinant protein hydrogel based on canine cartilage sequences, thereby reducing immunogenicity in canine subjects. In other embodiments, the matrix or controlled release formulation comprises a natural polymer such as alginate, chitosan, gelatin, hyaluronic acid, or4901-5416-2522.3Page 22 of 75 067505-000018WOPTcollagen. In other embodiments, the matrix or controlled release formulation comprises a synthetic polymer such as polyvinyl alcohol, polyethylene glycol, or poly(N- isopropylacrylamide), or another polymeric network. In yet another embodiment, the matrix or controlled release formulation may be based on microparticles for sustained or slow release.

[0105] In various embodiments, a composition or combination comprising recombinant coiled-coil protein derived from canine proteins and one, two, or more types of canine-specific immunomodulating agents is provided. In some embodiments, the recombinant coiled-coil protein is in a lyophilized or dry form. In some embodiments, the recombinant coiled-coil protein disposed in a biocompatible medium is in the form of a hydrogel. In various embodiments, coiled-coil protein hydrogel delivering bi- / multi-specific immune cell engagers and interferons are provided, wherein the bi- / multi-specific immune cell engagers comprises a first polypeptide capable of binding canine CD3 and a second polypeptide capable of binding an antigen associated with an autoimmune disease or a cancer. In various embodiments, the composition comprises coiled-coil protein hydrogel derived from canine cartilage protein, a bi- or multi-specific immune cell engager comprising a canine CD3-binding domain, and an interferon or another immune modulating protein, mini-protein, or small molecules. In some embodiments, two or more types of protein-based immunomodulating agents are encapsulated with the coiled-coil protein hydrogel. In some embodiments, the two or more types of immunomodulating agents are released from the protein hydrogel at a rate dependent on the density or concentration of the protein hydrogel. In some embodiments, the immune cell engagers, interferons, and / or other immune modulating proteins, mini-proteins, or small molecules are released from the coiled-coil protein hydrogel locally and in a sustained manner. In various embodiments, the composition is injected, implanted, or otherwise administered into the foci of cancer (aka: tumor), peritumoral tissue, or surgical cavity. In further embodiments, an administered composition is effective for killing or eliminating cancer cells locally. In further embodiments, the composition results in release of the one or more types of immunomodulating agents, thereby redirecting immune cells toward cancer cells.

[0106] In various embodiments, a recombinant protein comprises one or more self- association domains derived from canine cartilage proteins, preferably two or more thereof so as to flank, a flexible domain comprising a pseudorepeat of amino acids. In various embodiments, the flexible domain comprising the pseudorepeat of amino acids is intrinsically unstructured, is a pseudorepeat of 5-7 different amino acids (e.g., alanine, glutamic acid, glycine, proline, serine, and threonine), and comprises a length / quantity of 36-1,728 amino acids.4901-5416-2522.3Page 23 of 75 067505-000018WOPT

[0107] In various embodiments, the one or more self-association domains are structured, and wherein the structure comprises a coil. In various embodiments, a recombinant protein comprises two or more self-association domains and a flexible domain comprising a pseudorepeat of amino acids. In various embodiments, a self-association domain of a first recombinant protein molecule forms a reversible intermolecular interaction with a self- association domain of a second recombinant protein molecule, and vice versa. In some embodiments, a first self-association domain of a recombinant protein molecule forms a reversible intramolecular interaction with a second self-association domain of the recombinant protein molecule. In various embodiments, a recombinant protein molecule interacts with four additional recombinant protein molecules to form a pentameric unit between the self- association domains, connected by intermolecular interactions, intramolecular interactions, or a combination thereof.

[0108] In some embodiments, a recombinant protein, capable of forming coiled-coil protein hydrogel, comprises XTEN (a flexible domain) and self-associating helical (“coil”) end blocks, wherein the XTEN is flanked by the helical end blocks (i.e., an ABA type copolymer). In various aspects, the helical end blocks are peptide sequences derived from the N-terminal fragment of cartilage oligomeric matrix protein, (also called P domains). In various aspects, the helical end blocks between protein molecules spontaneously associate into homopentameric coiled-coil bundles to yield macroscopic gels.

[0109] In further embodiments, a protein, capable of forming coiled-coil protein hydrogel, comprises XTEN, canine cartilage oligomeric matrix protein fragment – preferably flanking XTEN, a signal peptide for improving secretion of proteins from mammalian cells, RGD peptide or another peptide promoting cell adhesion, and one or more purification tags.

[0110] In some embodiments, a signal peptide is included in the sequence of the protein. A signal peptide for increasing secretion of proteins may also be called as secretory signal sequence. Screening a library of signal peptides can be used to identify appropriate signal peptides for increasing expression (secretion) of a target protein. For example, a signal peptide has an amino acid sequence of METDTLLLWVLLLWVPGSTG (SEQ ID NO:1) or at least 95% sequence identity thereto. Further description of secretory signal sequences in mammalian expression systems is provided in U.S. Patent Nos. 8,241,870 and 8,883,450, WO / 2025 / 012213, and Humphreys et al. Protein Expression and Purification, vol.20, issue 2, pp 252:264 (2000), which are incorporated by reference herein.

[0111] In some embodiments, the canine cartilage oligomeric matrix protein fragment has an amino acid sequence of4901-5416-2522.3Page 24 of 75 067505-000018WOPTGPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA (SEQ ID NO:61), a truncated version such as GPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVME (SEQ ID NO:63). In some embodiments, the canine cartilage oligomeric matrix protein fragment has an amino acid sequence having at least 95%, 90%, 85%, or 80% sequence identity to GPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA (SEQ ID NO:61). In some embodiments, the canine cartilage oligomeric matrix protein fragment has an amino acid sequence having at least 95%, 90%, 85%, or 80% sequence identity to GPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVME (SEQ ID NO:63). In some embodiments, the canine cartilage oligomeric matrix protein fragment’s amino acid sequence is not APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA (SEQ ID NO:137), GPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMESDA (SEQ ID NO:138), APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:139), APQMLRELQEANAALQDVRELLRQQVKEITFLKNTVMESDA (SEQ ID NO:140), APQMLRELQEANAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:141), GPQMLRELQEANAALQDVRELLRQQVREITFLKNTVMESDA (SEQ ID NO:142), GPQMLRELQEANAALQDVRELLRQQVREITFLKNTVMESDAS (SEQ ID NO:143), or GPQMLRELQEANAALQDVRELLRQQVREITFLKNTVME (SEQ ID NO:144).

[0112] In some embodiments, one XTEN is flanked by a canine cartilage oligomeric matrix protein fragment of identical sequence on both ends. In some embodiments, one XTEN is flanked by a canine cartilage oligomeric matrix protein fragment of differing sequences.

[0113] In some embodiments, XTEN has an amino acid sequence of, or comprises a peptide having an amino acid sequence of, PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAP (SEQ ID NO:62), or at least 95%, 90%, 85%, or 80% sequence identity thereto. Further description of XTEN-based recombinant proteins, hydrogel thereof, linkers and additional motifs is provided in WO2024044721, which is incorporated by reference.

[0114] In some embodiments, a sequence for cell adhesion or a cell adhesion site comprises a peptide sequence of RGD. In some embodiments, a sequence for cell adhesion or a cell adhesion site has an amino acid sequence of GRGDS (SEQ ID NO:3). In some embodiments, a sequence for cell adhesion is included at both N-terminus and C-terminus relative to XTEN. In some embodiments, a sequence for cell adhesion is included at both ends of the flanked XTEN, i.e., from N- to C-terminus: a first cell adhesion sequence – a first4901-5416-2522.3Page 25 of 75 067505-000018WOPTcartilage oligomeric matrix protein fragment – XTEN – a second cartilage oligomeric matrix protein fragment – a second cell adhesion sequence. In some embodiments, the first and second cell adhesion sequences are identical. In some embodiments, the first and second cell adhesion sequences are different. In other embodiments, the recombinant protein composition does not comprise a cell adhesion peptide or cell adhesion motif.

[0115] In some embodiments, one or more tag sequences (or tag motifs) are included in the protein, including but not limited to a polyhistidine tag (having six (SEQ ID NO:60) or more histidine residues), a recognition motif such as LPXTG wherein X can be substituted with any amino acid (SEQ ID NO:136) (such as LPETG (SEQ ID NO:145)) – also referred to as a Sortag sequence, and / or a marker sequence. In various aspects, the inclusion of a marker sequence facilitates / ensures posttranslational cleavage of a signal sequence from the rest of the polypeptide. Preferably, the marker sequence is a short sequence, positioned between the signal sequence and the rest of the protein capable of forming hydrogel. In some embodiments, a marker sequence is one having an amino acid sequence of DYKDE (SEQ ID NO:2) – also referred to as a FLAG®-tag sequence. In some embodiments, a FLAG tag has an amino acid sequence of DYKDEGGS (SEQ ID NO:146).

[0116] Sortase A (SrtA) is an S. aureus transpeptidase, which is used as a bioorthogonal tool. Wild-type SrtA binds to the sorting motif “LPXTG” (where X is any amino acid) (SEQ ID NO:136) and cleaves between the Thr-Gly peptide bond. The Thr reacts with a Gly-Gly- Gly motif to form an LPXTGGG (SEQ ID NO:147) product, simultaneously displacing any sequence C-terminal to the sorting motif’s Thr. Wild-type SrtA has been evolved to yield two orthogonal sortases—eSrtA(2A9) and eSrtA(4S9) (respectively denoted 2A9 and 4S9) —that recognize with high specificity the sequences LAETG (SEQ ID NO:148) and LPESG (SEQ ID NO:149), respectively.

[0117] In various embodiments, the inclusion of a Sortag sequence (LPXTG (SEQ ID NO:136), such as LPETG (SEQ ID NO:145)) at the C-terminus of a protein allows for its binding / conjugation with another molecule that contains a sortase recognition motif (such as GGG or pentaglycine or diglycine) in the presence of sortase; thereby linking or immobilizing the protein with the molecule. In some embodiments, a protein disclosed herein based on XTEN flanked by cartilage oligomeric matrix protein fragment further comprises an RGD- containing motif and a LPXTGmotif, wherein X can be any amino acid (SEQ ID NO:136) such as E; and an immune cell engager molecule and / or IFN gamma may each be linked with a peptide comprising a sortase recognition motif (such as GGG or pentaglycine or diglycine); such that by adding a sortase in a mixture of the XTEN-based protein and the immune cell4901-5416-2522.3Page 26 of 75 067505-000018WOPTengager molecule and the IFN gamma, the immune cell engager molecule and / or the IFN gamma are linked to the XTEN-based protein.

[0118] In further embodiments, a protein disclosed herein based on XTEN flanked by cartilage oligomeric matrix protein fragment (roughly the ‘coiled coil protein’) further comprises a C-terminal sortase-recognition sequences (i.e., LPXTG, where X is any amino acid (SEQ ID NO:136)), which allows for site-specific modification with azide-functionalized polyglycine peptides (PolyG-azidoester) via chemoenzymatic transpeptidation. In some aspects, a cell adhesion peptide comprising RGD motif, a multi-specific (e.g., bispecific) immune cell engager molecule, and / or IFN gamma is functionalized with polyG-azidoester, such that they may chemoenzymatically conjugate with the coiled coil protein. In other embodiments, the cell adhesion peptide comprising RGD motif, the bispecific / multi-specific immune cell engager molecule, and / or IFN gamma is functionalized with a C-terminal sortase- recognition sequences (i.e., LPXTG, where X is any amino acid (SEQ ID NO:136)); whereas the coiled coil protein is functionalized with polyG-azidoester. Further description of sortase- mediated or polyG-azidoester-mediated conjugation and their synthesis is provided in US2022 / 0143210, which is incorporated by reference. In yet another embodiments, the bispecific / multi-specific immune cell engager molecule and / or the cytokine (e.g., IFN gamma) are functionalized with LPXTG, where X is any amino acid (SEQ ID NO:136), for conjugation with the recombinant protein.

[0119] In some embodiments, a multispecific antibody construct further includes an Fc, and / or a chemical moiety, such as a functional group for click chemistry or non-natural amino acids, for conjugation to a therapeutic moiety.

[0120] In some embodiments, an immunomodulating agent and / or a cytokine is fused with a self-associating domain, such that it may interact with a recombinant protein disclosed herein to yield macroscopic hydrogel.

[0121] In various embodiments, the protein comprising XTEN, cartilage oligomeric matrix protein fragment flanking the XTEN, a secretory signal sequence, and one or more of RGD peptide or another peptide promoting cell adhesion, linkers, and one or more tag sequences, forms (or self assembles into) hydrogel when reconstituted in an aqueous solution or biocompatible medium, such as phosphate-buffered saline. In some embodiment, the protein is resuspended in the reconstitution buffer with the immunomodulating agents (such as bi / multi-specific immune cell engager and IFN). In some embodiments, the suspension is vortexed, centrifuged, incubated at a higher temperature (e.g., 37 °C) for a brief period of time4901-5416-2522.3Page 27 of 75 067505-000018WOPT(e.g., 10 min), and gently rocked at a lower temperature (e.g., 4 °C) for a longer period of time (e.g., overnight) to encourage uniform gel formation.

[0122] In various embodiments, the recombinant protein is reconstituted in a biocompatible medium at a concentration of about 5% w / v in forming hydrogel. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 6% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 7% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 8% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 9% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 10% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 11% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 12% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 13% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 14% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at about 15% w / v. In some embodiments, the recombinant protein is reconstituted in a biocompatible medium at 15% w / v or more, In various embodiments, the protein is reconstituted at a concentration of at least 5% w / v in forming hydrogel. In various embodiments, the protein is reconstituted at 4.5%-6.5% w / v. In various embodiments, the protein is reconstituted at 6.5%-8.5% w / v. In various embodiments, the protein is reconstituted at 8.5%-11.5% w / v. In various embodiments, the protein is reconstituted at 11.5%-13.5% w / v. In various embodiments, the protein is reconstituted at 13.5%-15.5% w / v. In various embodiments, the protein is reconstituted at 15% w / v or more.

[0123] In various embodiments, a hydrogel composition comprising a hydrogel matrix formed of self-associated recombinant protein, an immune cell engager molecule, and a cytokine (preferably an interferon, or interferon gamma), is provided. In some embodiments, the immune cell engager molecule and / or the cytokine (e.g., interferon gamma) are non- covalently embedded / disposed / encapsulated in the hydrogel matrix. In some embodiments, the immune cell engager molecule and / or the cytokine (e.g., interferon gamma) are covalently attached to the hydrogel matrix or to the recombinant protein via a degradable linker.

[0124] In various embodiments, an immune cell engager molecule is a multispecific (including but not limited to bispecific) antibody construct. In various embodiments, an immune cell engager molecule (or multispecific antibody construct) comprises one or more of4901-5416-2522.3Page 28 of 75 067505-000018WOPTa first polypeptide capable of binding PD-L1 or B7-H3 or another tumor antigen, and one or more of a second polypeptide capable of binding canine CD3.

[0125] In various embodiments, the immune cell engager molecule (or multispecific antibody construct) further comprises a fragment crystallizable region (Fc), and optionally a linker between the Fc and at least one of the first polypeptide, between the Fc and the second polypeptide, between any two of the more first polypeptides, between any two of the more second polypeptides, or between at least one of the one or more first polypeptides and one of the one or more second polypeptides. In some embodiments, the Fc of a first polypeptide is an Fc “knob” sequence; and the Fc of a second polypeptide is an Fc “hole” sequence. Typically, one Fc CH3 domain contains engineered protuberance residues (“knob”) designed to fit into a cavity (“hole”) on the other Fc CH3 domain. The “hole” sequence heterodimerizes with the “knob” sequence (“knob-in-hole”). In some embodiments, a “knob” sequence has at least one contact residue replaced with an import residue having a larger side chain volume than the original residue; and a “hole” sequence at least one contact residue replaced with an import residue having a smaller side chain volume than the original residue. Volumes and accessible surface areas of each amino acid are described in A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972 and C. Chothia, J. Mol. Biol.105: 1-14, 1975. For example, import residues for the formation of a protuberance can be arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W); and preferably the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. As another example, import residues for the formation of a cavity can be alanine (A), serine (S), threonine (T) and valine (V); and preferably the original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. For example, a T366W mutation in CH3 domain for the “knob” / protuberance chain, and a T366S / L368A / Y407V mutation in CH3 domain for the “hole” / cavity chain. Additionally, the knob-in-hole configuration may be coupled further mutations to permit S-S disulfide linkage between the two chains. In other embodiments, no disulfide bonds are formed between the knob sequence and the hole sequence.

[0126] In various embodiments, two arms (or chains) of immunoglobulin heavy chain constant regions (e.g., Fc polypeptides) can be heterodimerized by creating “knobs-in-holes” (KiH) mutations in the CH3 domain. This structural feature in the polypeptide arms allows for assembly of two half antibodies (e.g., Fc heterodimer; and VH–CH and VL–CL domains). For example, a heteromultimer (including a heterodimer) may comprise two polypeptides, each comprising a CH3 domain, wherein the polypeptides meet at an engineered interface within4901-5416-2522.3Page 29 of 75 067505-000018WOPTthe CH3 domain, and the one polypeptide contains an engineered protuberance (“knob”) in the interface with at least one contact residue replaced with an import residue having a larger side chain volume than the original residue, and another polypeptide contains an engineered cavity (“hole”) in the interface with at least one contact residue replaced with an import residue having a smaller side chain volume than the original residue. In some embodiments, the engineered interface of a heteromultimer includes at least two protuberance-into-cavity mutant pairs. In various aspects wherein the protein / polypeptide is a heterodimer of the KiH configuration, each polypeptide of the canine CD3-binding molecule or of the multispecific antibody construct disclosed herein comprises one, and only one, of the two (or more) immunoglobulin heavy chain constant regions / chains (i.e. knob or hole).

[0127] In some embodiments, the multispecific antibody comprises, or its first and / or second polypeptide is in the form of a single-chain variable fragments (scFv). In some embodiments, the multispecific antibody comprises, or its first and / or second polypeptide is in the form of a Fab. In some embodiments, the multispecific antibody comprises, or its first and / or second polypeptide is in the form of a VHH domain. In some embodiments, the multispecific antibody comprises an scFv and a Fab, connected to a heterodimeric knob-in- hole Fc domain. In some embodiments, the multispecific antibody comprises, or at least one of its first and / or second polypeptide is capable of immunospecifically binding a tumor-associated antigen.

[0128] In various aspects, each polypeptide chain of a multispecific antibody construct comprises a VL domain and a VH domain. Interaction of two of the polypeptide chains will produce two VL-VH pairings, forming two epitope binding sites, i.e., a bivalent molecule. In various implementations, so long as the multispecific antibody construct form functional multispecificity, neither the VH or VL domain is constrained to any position within the polypeptide chain, i.e., restricted to the amino (N) or carboxy (C) terminus, nor are the domains restricted in their relative positions to one another, i.e., the VL domain may be N-terminal to the VH domain and vice-versa. Homodimerization of two polypeptide chains of an antibody specific for epitope A will result in the formation two VL-VH binding sites for epitope A, resulting in a bivalent monospecific antibody. Where the VL and VH domains are derived from antibodies specific for different antigens (A and B), formation of a functional bispecific construct requires the interaction of two different polypeptide chains, i.e., formation of a heterodimer. In some aspects, a multispecific antibody construct derived from antibodies specific for different antigen A and B may have a sequence from N-terminus to C-terminus:4901-5416-2522.3Page 30 of 75 067505-000018WOPTVLA—VHA—VHB—VLB, or in some instances short or flexible linkers are added: VLA— linker1—VHA—linker2—VHB—linker3—VLB.

[0129] One or more of the polypeptide chains of the multispecific antibody construct may optionally comprise an Fc domain or portion thereof (e.g. a CH2 domain, or CH3 domain). The Fc domain or portion thereof may be derived from any immunoglobulin isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE and IgM. In preferred embodiments, the Fc domain (or portion thereof) is derived from IgG. In specific embodiments, the IgG isotype is IgG1, IgG2, IgG3 or IgG4 or an allotype thereof. The Fc domain may be engineered into a polypeptide chain comprising the antigen-binding regions of the multispecific antibody construct in any position relative to other domains or portions of said polypeptide chain (e.g., the Fc domain, or portion thereof, may be c-terminal to both the VL and VH domains of the polypeptide of the chain; may be n-terminal to both the VL and VH domains; or may be N-terminal to one domain and c-terminal to another (i.e., between two domains of the polypeptide chain)). The Fc domains (if present) in the polypeptide chains of multispecific antibody construct preferentially dimerize, resulting in molecule that exhibits immunoglobulin- like properties, e.g., Fc-FcγR, interactions.

[0130] In some embodiments, a multispecific antibody construct is a bispecific antibody. In some embodiments, the multispecific antibody comprises one or more canine PD- L1-binding or canine B7-H3-binding polypeptides (or another polypeptide capable of binding an antigen other than CD3) and one or more canine CD3-binding polypeptides. In some embodiments, the polypeptides are produced with vectors encoding amino acid sequences as shown in FIG.4A-4L and 20A-20M.

[0131] In some embodiments, a multispecific antibody construct comprises a canine PD-L1-binding polypeptide and a canine CD3-binding polypeptide. In some embodiments, a multispecific antibody construct comprises an scFv derived from a canine PD-L1-binding antibody and an scFv derived from a canine CD3-binding antibody. In some embodiments, the canine PD-L1-binding scFv and the canine CD3-binding scFv are linked by a linker such as a GlySer linker. In some embodiments, a multispecific antibody construct comprises a canine PD-L1-binding scFv, a canine CD3-binding scFv, a signal peptide at the N-terminus, and a polyhistidine tag at the C-terminus. In some embodiments, a multispecific antibody construct comprises from N- to C-terminus: canine PD-L1-binding VL domain – canine PD-L1-binding VH domain – canine CD3-binding VH domain – canine CD3-binding VL domain, wherein each “—” may represent a GlySer linker. In other embodiments, the multispecific antibody construct may have the VH and VL domains of the two antigens in a reversed sequence. In4901-5416-2522.3Page 31 of 75 067505-000018WOPTsome embodiments, a multispecific antibody construct comprises an scFv derived from a canine PD-L1-binding antibody and a Fab derived from a canine CD3-binding antibody. In some embodiments, a multispecific antibody construct comprises a Fab derived from a canine PD-L1-binding antibody and an scFv derived from a canine CD3-binding antibody. In some embodiments, a multispecific antibody construct comprises a Fab derived from a canine PD- L1-binding antibody and a Fab derived from a canine CD3-binding antibody.

[0132] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.2A12, and the canine PD-L1-binding polypeptide is from the antibody of clone 4F9. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.2A12, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 4F9.

[0133] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.6F9, and the canine PD-L1-binding polypeptide is from the antibody of clone 4F9. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.6F9, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 4F9.

[0134] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.8D4, and the canine PD-L1-binding polypeptide is from the antibody of clone 4F9. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.8D4, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 4F9.

[0135] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.3G9, and the canine PD-L1-binding polypeptide is from the antibody of clone 4F9. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.3G9, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 4F9.4901-5416-2522.3Page 32 of 75 067505-000018WOPT

[0136] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.5F11, and the canine PD-L1-binding polypeptide is from the antibody of clone 4F9. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.5F11, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 4F9.

[0137] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.6B3, and the canine PD-L1-binding polypeptide is from the antibody of clone 4F9. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.6B3, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 4F9.

[0138] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.9C5, and the canine PD-L1-binding polypeptide is from the antibody of clone 4F9. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.9C5, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 4F9.

[0139] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.2A12, and the canine PD-L1-binding polypeptide is from the antibody of clone 5F12. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.2A12, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 5F12.

[0140] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.6F9, and the canine PD-L1-binding polypeptide is from the antibody of clone 5F12. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.6F9, and the canine PD-L1-binding4901-5416-2522.3Page 33 of 75 067505-000018WOPTpolypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 5F12.

[0141] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.8D4, and the canine PD-L1-binding polypeptide is from the antibody of clone 5F12. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.8D4, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 5F12.

[0142] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.3G9, and the canine PD-L1-binding polypeptide is from the antibody of clone 5F12. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.3G9, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 5F12.

[0143] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.5F11, and the canine PD-L1-binding polypeptide is from the antibody of clone 5F12. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.5F11, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 5F12.

[0144] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.6B3, and the canine PD-L1-binding polypeptide is from the antibody of clone 5F12. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.6B3, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 5F12.

[0145] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.9C5, and the canine PD-L1-binding polypeptide is from the antibody of clone 5F12. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95%4901-5416-2522.3Page 34 of 75 067505-000018WOPTsequence identity to that of the antibody of clone CA17.9C5, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone 5F12.

[0146] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.2A12, and the canine PD-L1-binding polypeptide is from atezolizumab. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.2A12, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of atezolizumab.

[0147] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.6F9, and the canine PD-L1-binding polypeptide is from atezolizumab. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.6F9, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of atezolizumab.

[0148] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.8D4, and the canine PD-L1-binding polypeptide is from atezolizumab. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.8D4, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of atezolizumab.

[0149] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.3G9, and the canine PD-L1-binding polypeptide is from atezolizumab. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.3G9, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of atezolizumab.

[0150] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.5F11, and the canine PD-L1-binding polypeptide is from atezolizumab. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.5F11, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of atezolizumab.4901-5416-2522.3Page 35 of 75 067505-000018WOPT

[0151] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.6B3, and the canine PD-L1-binding polypeptide is from atezolizumab. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.6B3, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of atezolizumab.

[0152] In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is from the antibody of clone CA17.9C5, and the canine PD-L1-binding polypeptide is from atezolizumab. In some embodiments of a multispecific antibody, the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of the antibody of clone CA17.9C5, and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of atezolizumab.

[0153] Further description of PD-L1 binding peptides and bispecific or multispecific antibody constructs is provided in US20240287147, and exemplary PD-L1 antibodies capable of binding canine PD-L1 – such as clones 4F9 and 5F12 – are provided in US Pat. No. 10,550,194, which are incorporated by reference. Canine PD-L1 antibody of clone 4F9 comprises a VH domain comprising an amino acid sequence of SEQ ID NO:13, and a VL domain comprising an amino acid sequence of SEQ ID NO:12. Canine PD-L1 antibody of clone 5F12 comprises a VH domain comprising an amino acid sequence of SEQ ID NO:15, and a VL domain comprising an amino acid sequence of SEQ ID NO:14. In yet another embodiment, a PD-L1-binding peptide may comprise the VH and / or VL of atezolizumab, a fully humanized, IgG1 monoclonal antibody that blocks the interaction of PD-L1 with both PD-1 and B7.1.

[0154] In some embodiments, a multispecific antibody construct comprises a canine B7-H3-binding polypeptide and a canine CD3-binding polypeptide. In some embodiments, a multispecific antibody construct comprises an scFv derived from a canine B7-H3 antibody and an scFv derived from a canine CD3-binding antibody. In some embodiments, the canine B7- H3-binding scFv and the canine CD3-binding scFv are linked by a linker such as a GlySer linker. In some embodiments, a multispecific antibody construct comprises a canine B7-H3- binding scFv, a canine CD3-binding scFv, a signal peptide at the N-terminus, and a polyhistidine tag at the C-terminus. In some embodiments, a multispecific antibody construct comprises from N- to C-terminus: canine B7-H3-binding VL domain – canine B7-H3-binding VH domain – canine CD3-binding VH domain – canine CD3-binding VL domain, wherein each “—” may represent a GlySer linker. In other embodiments, the multispecific antibody4901-5416-2522.3Page 36 of 75 067505-000018WOPTconstruct may have the VH and VL domains of the two antigens in a reversed sequence. In some embodiments, a multispecific antibody construct comprises an scFv derived from a canine B7-H3-binding antibody and a Fab derived from a canine CD3-binding antibody. In some embodiments, a multispecific antibody construct comprises a Fab derived from a canine B7-H3-binding antibody and an scFv derived from a canine CD3-binding antibody. In some embodiments, a multispecific antibody construct comprises a Fab derived from a canine B7- H3-binding antibody and a Fab derived from a canine CD3-binding antibody.

[0155] In some embodiments, a B7-H3-binding peptide may comprise the VH and / or VL of enoblituzumab (also known as MGA271), an Fc optimized humanized IgG1 monoclonal antibody that binds to B7-H3 (CD276).

[0156] In some embodiments of a multispecific antibody, the canine B7-H3-binding polypeptide is from enoblituzumab, and the canine CD3-binding polypeptide is from the antibody of clone CA17.9C5, CA17.2A12, CA17.8D4, CA17.6F9, CA17.3G9, CA17.5F11, or CA17.6B3. In some embodiments of a multispecific antibody, the canine B7-H3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of atezolizumab; and the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of respective antibody of clone CA17.9C5, CA17.2A12, CA17.8D4, CA17.6F9, CA17.3G9, CA17.5F11, or CA17.6B3.

[0157] In some embodiments, a multispecific antibody construct comprises a canine EphA2-binding polypeptide and a canine CD3-binding polypeptide. In some embodiments, a multispecific antibody construct comprises an scFv derived from a canine EphA2 antibody and an scFv derived from a canine CD3-binding antibody. In some embodiments, the canine EphA2-binding scFv and the canine CD3-binding scFv are linked by a linker such as a GlySer linker. In some embodiments, a multispecific antibody construct comprises a canine EphA2- binding scFv, a canine CD3-binding scFv, a signal peptide at the N-terminus, and a polyhistidine tag at the C-terminus. In some embodiments, a multispecific antibody construct comprises from N- to C-terminus: canine EphA2-binding VL domain – canine EphA2-binding VH domain – canine CD3-binding VH domain – canine CD3-binding VL domain, wherein each “—” may represent a GlySer linker. In other embodiments, the multispecific antibody construct may have the VH and VL domains of the two antigens in a reversed sequence. In some embodiments, a multispecific antibody construct comprises an scFv derived from a canine EphA2-binding antibody and a Fab derived from a canine CD3-binding antibody. In some embodiments, a multispecific antibody construct comprises a Fab derived from a canine EphA2-binding antibody and an scFv derived from a canine CD3-binding antibody. In some4901-5416-2522.3Page 37 of 75 067505-000018WOPTembodiments, a multispecific antibody construct comprises a Fab derived from a canine EphA2-binding antibody and a Fab derived from a canine CD3-binding antibody.

[0158] In some embodiments, a canine EphA2-binding peptide may comprise a VH having an amino acid sequence of SEQ ID NO:129 or comprise the CDR1, CDR2, and CDR3 of the EphA2-binding VH; and / or the canine EphA2-binding peptide may comprises a VL having an amino acid sequence of SEQ ID NO:128 or comprise the CDR1, CDR2, and CDR3 of the EphA2-binding VL.

[0159] In some embodiments of a multispecific antibody, the canine EphA2-binding polypeptide comprises a VH domain having an amino acid sequence of SEQ ID NO:129 and / or a VL domain having an amino acid sequence of SEQ ID NO:128; and the canine CD3-binding polypeptide is from the antibody of clone CA17.9C5, CA17.2A12, CA17.8D4, CA17.6F9, CA17.3G9, CA17.5F11, or CA17.6B3. In some embodiments of a multispecific antibody, the canine EphA2-binding polypeptide comprises a VH domain having at least 95% sequence identity to that of SEQ ID NO:129, and / or a VL domain having at least 95% sequence identity to that of SEQ ID NO:128; and the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of respective antibody of clone CA17.9C5, CA17.2A12, CA17.8D4, CA17.6F9, CA17.3G9, CA17.5F11, or CA17.6B3.

[0160] In some embodiments, a multispecific antibody construct comprises a CEACAM5-binding polypeptide and a canine CD3-binding polypeptide. In some embodiments, a multispecific antibody construct comprises an scFv derived from a CEACAM5 antibody and an scFv derived from a canine CD3-binding antibody. In some embodiments, the CEACAM5-binding scFv and the canine CD3-binding scFv are linked by a linker such as a GlySer linker. In some embodiments, a multispecific antibody construct comprises a CEACAM5-binding scFv, a canine CD3-binding scFv, a signal peptide at the N-terminus, and a polyhistidine tag at the C-terminus. In some embodiments, a multispecific antibody construct comprises from N- to C-terminus: CEACAM5-binding VL domain – CEACAM5-binding VH domain – canine CD3-binding VH domain – canine CD3-binding VL domain, wherein each “—” may represent a GlySer linker. In other embodiments, the multispecific antibody construct may have the VH and VL domains of the two antigens in a reversed sequence. In some embodiments, a multispecific antibody construct comprises an scFv derived from a CEACAM5-binding antibody and a Fab derived from a canine CD3-binding antibody. In some embodiments, a multispecific antibody construct comprises a Fab derived from a CEACAM5- binding antibody and an scFv derived from a canine CD3-binding antibody. In some4901-5416-2522.3Page 38 of 75 067505-000018WOPTembodiments, a multispecific antibody construct comprises a Fab derived from a CEACAM5- binding antibody and a Fab derived from a canine CD3-binding antibody.

[0161] In some embodiments, a CEACAM5-binding peptide may comprise a VH having an amino acid sequence of SEQ ID NO:132 or 135 or comprise the CDRs of the CEACAM5-binding VH; and / or the CEACAM5-binding peptide may comprise a VL having an amino acid sequence of SEQ ID NO:131 or 134 or comprise the CDRs of the CEACAM5- binding VL.

[0162] In some embodiments of a multispecific antibody, the CEACAM5-binding polypeptide comprises a VH domain having an amino acid sequence of SEQ ID NO:132 and / or a VL domain having an amino acid sequence of SEQ ID NO:131; and the canine CD3-binding polypeptide is from the antibody of clone CA17.9C5, CA17.2A12, CA17.8D4, CA17.6F9, CA17.3G9, CA17.5F11, or CA17.6B3. In some embodiments of a multispecific antibody, the CEACAM5-binding polypeptide comprises a VH domain having an amino acid sequence of SEQ ID NO:135 and / or a VL domain having an amino acid sequence of SEQ ID NO:134; and the canine CD3-binding polypeptide is from the antibody of clone CA17.9C5, CA17.2A12, CA17.8D4, CA17.6F9, CA17.3G9, CA17.5F11, or CA17.6B3. In some embodiments of a multispecific antibody, the CEACAM5-binding polypeptide comprises a VH domain having at least 95% sequence identity to that of SEQ ID NO:132 or 135, and / or a VL domain having at least 95% sequence identity to that of SEQ ID NO:131 or 134; and the canine CD3-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of respective antibody of clone CA17.9C5, CA17.2A12, CA17.8D4, CA17.6F9, CA17.3G9, CA17.5F11, or CA17.6B3.

[0163] In some embodiments, a multispecific antibody construct comprises a canine B7-H3-binding polypeptide, a canine CD3-binding polypeptide, and a PD-L1-binding polypeptide. In some embodiments, a multispecific antibody construct comprises three linked scFv, wherein an scFv derived from a BH-H3 antibody is linked to an scFv derived from a CD3 antibody, and the latter is linked to an scFv derived from PD-L1 antibody; for example, SCRI- 002320 in figure 14.

[0164] In some embodiments a multispecific antibody construct comprises a canine CD3-binding polypeptide and a ROR1-binding polypeptide. In some embodiments, a multispecific antibody construct comprises an scFv derived from a CD3 antibody linked to a VHH derived from an ROR1 antibody; for example, SCRI-002324 in figure 14.

[0165] In some embodiments, a multispecific antibody construct comprises a canine CD3-binding polypeptide, a PD-L1-binding polypeptide, and a ROR1-binding polypeptide. In4901-5416-2522.3Page 39 of 75 067505-000018WOPTsome embodiments a multispecific antibody construct comprises heterodimeric key-in-hole IgGs, wherein one IgG chain is linked to a CD3-binding polypeptide, the other IgG chain is linked to a PD-L1-binding polypeptide, and at least one of the two IgG chains (preferably both IgG chains) are linked to an ROR1-binding polypeptide. In this construct, the CD3-binding polypeptide may be an scFv. In this construct, the CD3-binding polypeptide may be a Fab. In this construct, the PD-L1-binding polypeptide may be an scFv. In this construct, the PD-L1- binding polypeptide may be a Fab. In this construct, the ROR1-binding polypeptide may be a VHH. See examples like SCRI-002325 and SCRI-002326 in figure 14.

[0166] Exemplary ROR1 antibodies or VHH derived therefrom are shown in at least US Pat. No.12,122,830.

[0167] In some embodiments, a multispecific antibody construct comprises a CD28- binding (e.g. canine CD28-binding) polypeptide and a canine PD-L1-binding polypeptide. In some embodiments, a multispecific antibody construct comprises an scFv derived from a CD28-binding antibody and an scFv derived from a canine PD-L1-binding antibody. In some embodiments, the CD28-binding scFv and the canine PD-L1-binding scFv are linked by a linker such as a GlySer linker. In some embodiments, a multispecific antibody construct comprises a CD28-binding scFv, a canine PD-L1-binding scFv, a signal peptide at the N- terminus, and a polyhistidine tag at the C-terminus. In some embodiments, a multispecific antibody construct comprises from N- to C-terminus: CD28-binding VL domain – CD28- binding VH domain – canine PD-L1-binding VH domain – canine PD-L1-binding VL domain, wherein each “—” may represent a GlySer linker. In other embodiments, the multispecific antibody construct may have the VH and VL domains of the two antigens in a reversed sequence. In some embodiments, a multispecific antibody construct comprises an scFv derived from a CD28-binding antibody and a Fab derived from a canine PD-L1-binding antibody. In some embodiments, a multispecific antibody construct comprises a Fab derived from a CD28- binding antibody and an scFv derived from a canine PD-L1-binding antibody. In some embodiments, a multispecific antibody construct comprises a Fab derived from a CD28- binding antibody and a Fab derived from a canine PD-L1-binding antibody.

[0168] Exemplary canine CD28-binding antibodies are described at least by Graves et al. in Transplantation 2011 Apr 27;91(8):833–840. In some embodiments, a canine CD28- binding polypeptide disclosed herein may comprise a VH having an amino acid sequence of that of a canine CD28 antibody; and / or the canine CD28-binding polypeptide may comprise a VL having an amino acid sequence of that of a canine CD28 antibody. In some embodiments,4901-5416-2522.3Page 40 of 75 067505-000018WOPTexemplary VH and VL sequences of canine CD28 are seen in the sequences in Figures 20F and 20G.

[0169] In some embodiments, a multispecific antibody construct comprises a CD40- binding polypeptide and a canine PD-L1-binding polypeptide. In some embodiments, a CD40- binding polypeptide comprises CD40L, or two or more copies of CD40L. In some embodiments, a multispecific antibody construct comprises a first polypeptide comprising three CD40L and an scFv derived from a canine PD-L1-binding antibody. In some embodiments, the one or more CD40L and the canine PD-L1-binding scFv are linked by a linker such as a GlySer linker. In some embodiments, a multispecific antibody construct comprises one or more CD40L, a canine PD-L1-binding scFv, a signal peptide at the N- terminus, and a polyhistidine tag at the C-terminus. In some embodiments, a multispecific antibody construct comprises from N- to C-terminus: CD40L – CD40L – CD40L – canine PD- L1-binding VL domain, wherein each “—” may represent a GlySer linker. In some embodiments, a multispecific antibody construct comprises one or more CD40L and a Fab derived from a canine PD-L1-binding antibody.

[0170] In some embodiments, a CD40L peptide comprises an amino acid sequence of SEQ ID NO:121. In some embodiments, two or more linked CD40L is connected to a first IgG Fc domain, and the PD-L1-binding polypeptide is connected to a second IgG Fc domain, wherein the first and the second IgG Fc domains form a knob-in-hole heterodimer.

[0171] In some embodiments of a multispecific antibody, the CD40-binding polypeptide comprises three CD40L; and the canine PD-L1-binding polypeptide is from the antibody of clone 4F9 or 5F12 or from atezolizumab. In some embodiments of a multispecific antibody, the CD40-binding polypeptide comprises two or more copies each having at least 95% sequence identity to that of SEQ ID NO:121; and the canine PD-L1-binding polypeptide is an scFv, Fab, or VHH having at least 95% sequence identity to that of respective antibody of clone 4F9 or 5F12 or that of atezolizumab.

[0172] In some embodiments, a multi-specific antibody construct and / or the cytokine are covalently attached to the recombinant protein via degradable linkers. In some embodiments, a multi-specific antibody construct and / or the cytokine are covalently attached to the recombinant protein via non-degradable linkers. In some embodiments, a multi-specific antibody construct and / or the cytokine are covalently attached to the recombinant protein via protease-degradable linker. In some embodiments, a multi-specific antibody construct and / or a cytokine are linked to the recombinant protein via linkers of different degradation rates.4901-5416-2522.3Page 41 of 75 067505-000018WOPT

[0173] In various embodiments, the multispecific antibody construct (also referred to as immune cell engager molecule) and / or the cytokine (preferably an interferon, or interferon gamma) are released from the hydrogel composition or controlled release or timed release formulation over a time span of at least 5 days. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 10 days. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 1 week. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 2 weeks. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 3 week. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 4 weeks. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 5 weeks. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 6 weeks. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 7 weeks. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 8 weeks. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 9 weeks. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over at least 10 weeks. In some embodiments, the multispecific antibody construct and the cytokine (if present) are released from the hydrogel composition or controlled release or timed release formulation over 10 weeks or longer. In some embodiments, about 1%-18% or no more than about 20% of the immune cell engager molecule and about 1%-18% of the interferon of the initial hydrogel composition or controlled release or timed release formulation are released from the hydrogel composition in about 1 day. In some embodiments, about 15%-20% or no more than about 20% of the immune cell engager4901-5416-2522.3Page 42 of 75 067505-000018WOPTmolecule and about 15%-20% of the interferon of the initial hydrogel composition or controlled release or timed release formulation are released in about 1 day from the hydrogel composition comprising about 5% w / v of the recombinant protein. In some embodiments, about 10%-20% of the immune cell engager molecule and about 10%-20% of the interferon of the initial hydrogel composition or controlled release or timed release formulation are released in about 1 day from the hydrogel composition comprising about 10% w / v of the recombinant protein. In some embodiments, about 5%-12% of the immune cell engager molecule and about 5%- 12% of the interferon of the initial hydrogel composition or controlled release or timed release formulation are released in about 1 day from the hydrogel composition comprising about 12.5% w / v of the recombinant protein. In some embodiments, cumulatively about 15%-25% of the immune cell engager molecule and about 15%-25% of the interferon of the initial hydrogel composition or controlled release or timed release formulation are released in about 5 days from the hydrogel composition or controlled release or timed release formulation comprising about 5% w / v of the recombinant protein. In some embodiments, cumulatively about 10%- 30% of the immune cell engager molecule and about 20%-30% of the interferon of the initial hydrogel composition or controlled release or timed release formulation are released in about 9 days from the hydrogel composition comprising about 5% w / v of the recombinant protein. Generally, release is slower from a hydrogel composition or controlled release or timed release formulation comprising a higher concentration of the polymeric constituent, compared to a lower concentration of the constituent; and release is slow from a hydrogel composition or controlled release or timed release formulation wherein the immune cell engager molecule and / or the cytokine are covalently linked to the recombinant protein, compared to noncovalently or physically encapsulated.

[0174] In some embodiments, the composition further comprises one or more cell types encapsulated in the coiled-coil protein hydrogel or the controlled release or timed release formulation. For example, fibroblasts, hepatocytes, embryonic kidney (HEK), embryonic stem- cell-derived cardiomyocytes, induced pluripotent stem cells, or other cells may be encapsulated in the composition.

[0175] In some embodiments, the composition does not include one or more cell types. For example, a coiled-coil protein hydrogel encapsulating or admixed with bi- / multi-specific immune cell engagers and interferons does not encapsulate or contain cells.

[0176] In some embodiments, a biocompatible medium includes suitable carriers, diluents and / or excipients. In some embodiments, a biocompatible medium includes one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as4901-5416-2522.3Page 43 of 75 067505-000018WOPTwell as any combination thereof. In some cases, it includes isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition. In some embodiments, suitable carriers include: (1) phosphate buffered sale or Dulbecco’s phosphate buffered saline, pH about 7.4, (2) 0.9% saline (0.9% w / v sodium chloride (NaCl)), and (3) 5% (w / v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as TWEEN 20®.

[0177] Multispecific antibody constructs are also provided, which may be used without a hydrogel formed from the recombinant protein, and the multispecific antibody constructs include: one or more of a first polypeptide capable of binding canine CD3, and one or more of a second polypeptide capable of binding an antigen associated with a disease. In some embodiments, when the antigen associated with a disease comprises B7-H3, the first polypeptide capable of binding canine CD3 comprises complementarity-determining regions (CDRs) of anti-dog CD3 antibody of clone CA17.8D4, CA17.6F9, or both, or does not comprise CDRs of CA17.2A12.

[0178] In some embodiments, a multispecific antibody construct includes: one or more of a first polypeptide capable of binding canine CD3 comprising CDRs of anti-dog CD3 antibody clone CA17.8D4 or CA17.6F9 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CA17.8D4 or CA17.6F9, and one or more of a second polypeptide capable of binding an antigen associated with a disease, such as canine cancer or canine autoimmune disease. Treatment Methods

[0179] The CD3-binding molecules of the present invention have particular utility in the treatment of cancers associated with CD3 expression and in the treatment of autoimmune disease and other inflammatory disorders. These uses can involve the formation of a complex between CD3 and an antibody that binds specifically to CD3. Without being bound by theory, anti-CD3 antibody can bind to CD3 through the extracellular domain of CD3 and may then be internalized inside of a living normal or cancer cell.

[0180] The antibodies and antigen-binding fragments of the present disclosure can bind to CD3 present on the surface of T cells; and the CD3-binding molecule can be used in the context of a multispecific antibody construct to direct T-cells to a tumor cell, whereby the T- cells can then kill the tumor cell. In various embodiments, the composition is for use in treatment of solid tumors, wherein the composition comprises coiled-coil protein hydrogel encapsulating a multi-specific (including but not limited to bispecific) antibody construct that effectuates as an immune cell engager and a cytokine such as interferons. In some embodiments, a method of treating a canine subject with solid tumor such as brain tumor,4901-5416-2522.3Page 44 of 75 067505-000018WOPTmelanoma (such as oral melanoma), or soft tissue sarcoma includes administering to the subject a composition comprising coiled-coil protein hydrogel, multi-specific immune cell engagers, and interferons, wherein the multi-specific immune cell engagers and interferons are admixed with or encapsulated in the coiled-coil protein hydrogel.

[0181] The second antigen or epitope for a multispecific antibody construct is preferably a tumor antigen expressed on a tumor cell. Such tumor cells may be from cancers, for example, breast cancer, prostate cancer, gastric cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma, and leukemia. The additional antigens or epitopes are preferably cell surface tumor antigens or epitopes (such as: 17-1A, A33, adult erythrocyte primary endoderm I antigen, alpha fetoprotein, an envelope antigen of an RNA tumor virus, bladder tumor oncofetal antigen, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, Burkitt's lymphoma antigen-38.13, CA125, CD18, CD19, human B-lymphoma antigen-CD20, CD22, CD33, CD44, CD52, CEA, CO17-1A, CTA-1, CTLA-4, epidermal growth factor receptor, Ep- CAM, EphA2, fetal erythrocyte I antigen, fibrosarcoma antigen, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, GICA 19-9, gp IIIb / IIIa, gp72, HER1, HER-2 / neu, HER3, HER4, high molecular weight melanoma antigen, HLA-DR antigen, human leukemia T cell antigen-Gp37, human lung carcinoma antigen L20, human lung carcinoma antigen L6, human milk fat globule antigen, IgE, KS 1 / 4 pan-carcinoma antigen, LEA, lung adenocarcinoma F3 antigen, malignant human lymphocyte antigen-APO-1, melanoma antigen gp75, melanoma-associated antigen p97, neoglycoprotein, nuC242, polymorphic epithelial mucin antigen, prostate specific antigen, prostate specific membrane antigen, prostatic acid phosphate, SK-1 antigen, TAG-72, T-antigen, tumor antigen CA125, tumor antigen MUC1, tumor-specific transplantation type of cell-surface antigen, vascular endothelial growth factor, vascular endothelial growth factor-receptor, and αvβ3). Alternatively, such additional antigens or epitopes may be associated with a pathogen (such as: hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, small pox, Epstein Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), viral miningitis, viral encephalitis, dengue, small pox; mycobacteria rickettsia,4901-5416-2522.3Page 45 of 75 067505-000018WOPTmycoplasma, Neisseria, S. pneumonia, Borrelia burgdorferi, Bacillus anthraces, Streptococcus, Staphylococcus, Mycobacterium, tetanus, pertissus, cholera, plague, diptheria, chlamydia, and legionella; leishmania, kokzidioa, trypanosoma or malaria; chlamydia and rickettsia. In some embodiments, PD-L1 may be used as the “second” epitope. In some embodiments, B7-H3 may be used as the “second” epitope. In some embodiments, CD28 may be used as the “second” epitope. In some embodiments, CD40 may be used as the “second” epitope. In some embodiments, EphA2 may be used as the “second” epitope. In some embodiments, CEACAM5 may be used as the “second” epitope.

[0182] In some embodiments, the composition is for use in treating recurrent tumors, e.g., in a canine subject having had surgery. In some embodiments, the canine subject has recurrent tumors after a prior treatment. In some embodiments, the canine subject has tumor and the treatment method directly injects the composition into tumor parenchyma, without undergoing surgery.

[0183] In some embodiments, the composition is for use in treating local cancers, such as small melanomas, retinoblastoma, which may otherwise require surgery. In some embodiments, the composition is for use in treating local cancer such as parameningeal sarcomas, pelvic sarcomas, ocular tumors, which are in sites that preclude complete surgical resection. In some embodiments, the composition is for use in treating cancers such as optic gliomas, para-tracheal or para-bronchial lung cancer, which anatomically compress critical structures (e.g., ureter compression, urethral compression, esophageal obstruction). In some embodiments, the composition is administered in the tumor cavity or peritumoral tissue during / after surgical resection.

[0184] Further embodiments provide the composition is for use in killing of cancer cells. In further embodiments, the composition is for use in recruiting and / or modulating immune cells, so as to inhibit cancer cells.

[0185] In some embodiments, the cancer is a PD-L1-expressing cancer. In some embodiments, the cancer is a B7-H3-expressing cancer. In some embodiments, the cancer comprises brain tumor, or the cancer cell comprises medulloblastoma cell.

[0186] In some embodiments, the cancer in the treatment method comprises glioblastoma, or the cancer cell comprises glioblastoma cell. In some embodiments, the cancer comprises melanoma, or oral melanoma. In some embodiments, the cancer comprises soft tissue sarcoma. In some embodiments, the cancer comprises lung cancer, hematological cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer, ovarian cancer, renal cancer, uterine cancer, bladder cancer, kidney cancer, melanoma, thyroid cancer, myeloid leukemia,4901-5416-2522.3Page 46 of 75 067505-000018WOPTmantle cell lymphoma, or multiple myeloma. In some embodiments, the composition is for use in treating a canine cancer, such as lymphoma, nasal or anal cancers, mammary carcinoma, recurrent solid tumors or solid or brain tumors. In some embodiments, the canine cancer poses a high risk of recurrence or metastatic spread.

[0187] The multispecific antibody constructs disclosed herein may be administered to a patient in one or more unit doses of typically 0.0001 mg / kg to 100 mg / kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg or 0.01 to 0.10 mg / kg of the patient's body weight.

[0188] In some embodiments, the composition is for use in treatment of overgrowth disorders such as neurofibromatosis, tuberous sclerosis, and the like. In some embodiments, a method of treating neurofibromatosis, tuberous sclerosis, or another overgrowth disorder in a subject is provided, which includes administering to the subject a composition comprising coiled-coil protein hydrogel, a multi-specific (e.g., bi-specific) immune cell engager, and an interferon, wherein the immune cell engager and the interferon are admixed with, encapsulated in, or otherwise delivered by the coiled-coil protein hydrogel.

[0189] In some embodiments, the treatment methods with the composition replaces surgery. In some embodiments, the composition is administered at a parameningeal site, pelvis, or intravitreal site of a subject. In some embodiments, the treatment methods reduce the risk of local or distant recurrence of tumors that have been resected. In some embodiments, the treatment methods reduce the risk of metastatic spread, and for palliation (reduce size of tumors that cause discomfort when patient desires comfort rather than cure).

[0190] In some embodiments, the subject is an adult canine. In some embodiments, the subject is a pediatric dog. Canine CD3 antibodies and uses thereof

[0191] The present disclosure encompasses compositions, including pharmaceutical compositions, comprising anti-CD3 antibodies, polypeptides derived from anti-CD3 antibodies, polynucleotides comprising sequence encoding anti-CD3 antibodies, and other agents as described herein. The disclosure further provides for the anti-CD3 antibodies, polypeptides derived from anti-CD3 antibodies, or polynucleotides bound to a macromolecule such as any insoluble, solid support matrix used in diagnostic, screening, purification, or treatment procedures. In various embodiments, the CD3-binding molecules or anti-CD34901-5416-2522.3Page 47 of 75 067505-000018WOPTantibodies disclosed herein are characterized but not limited by one or more of: an ability to specifically bind canine CD3 as endogenously expressed on the surface of a normal canine T cell; an ability to specifically bind canine CD3 as endogenously expressed on the surface of a canine leukemic T cell; an ability to neutralize (i.e., block or interfere with binding) the formation of the CD3 complex; an ability to neutralize the formation of the TCR complex; an ability to modulate (either antagonistically or agonistically) signaling by the TCR complex; an ability to bind the Fc receptor; and / or an ability to bind to a portion of CD3 that is exposed on the surface of a living cell in vitro or in vivo; an ability to bind to a portion of CD3 that is exposed on the surface of a living cancer cell.

[0192] The present disclosure provides any of the following (or compositions, including pharmaceutical compositions, comprising any of the following): (a) an antibody produced by the host cell or its progeny; (b) a humanized form of such an antibody; (c) an antibody comprising one or more of the light chain and / or heavy chain variable regions of such an antibody; (d) a chimeric antibody comprising variable regions homologous or derived from variable regions of a heavy chain and a light chain of such an antibody, and constant regions homologous or derived from constant regions of a heavy chain and a light chain of a human antibody; (e) an antibody comprising one or more of the light chain and / or heavy chain CDRs (at least one, two, three, four, five, or six) of such an antibody; (f) an antibody comprising a heavy and / or a light chain of such an antibody; and (g) a human antibody that is equivalent to such an antibody. Other embodiments include antibodies that have at least two, three, four, five, or six CDR(s) that are substantially homologous to at least two, three, four, five or six CDRs of an antibody produced from a hybridoma deposited as identified herein, or derived from such an antibody.

[0193] Various embodiments provide a canine CD3-binding molecule, which includes: a CD3-specific VL domain and a CD3-specific VH domain of one anti-dog CD3 antibody selected from clone CD17.2A12, CA17.6F9, or CA17.8D4 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CD17.2A12, CA17.6F9, or CA17.8D4, optionally the variant of the clone CD17.6F9 comprising an amino acid substitution in the CD3- specific VL domain at the N-terminus from Ile (I) to Asp (D) or comprising an amino acid sequence of SEQ ID NO:11.

[0194] Additional embodiments provide a canine CD3-binding molecule, which includes: a CD3-specific VL domain and / or a CD3-specific VH domain of clone CD17.5F11 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CD17.5F11.4901-5416-2522.3Page 48 of 75 067505-000018WOPTIn some embodiments, a canine CD3-binding molecule includes a CD3-specific VL domain and a CD3-specific VH domain of clone CD17.5F11.

[0195] Amino acid sequence of the VL domain of clone CD17.5F11: DILLTQSPATLSVTPGETVSLSCRASQSIYKNLHWYQQKSHRSPRLLIKYASDSISGIPS RFTGSGSGTDYTLSINSVKPEDEGIYYCLQGYSTPWTFGGGTKLEIK (SEQ ID NO:80), wherein the three CDRs (as determined by the IMGT numbering system) are in bolded italics.

[0196] Amino acid sequence of the VH domain of clone CD17.5F11: EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSHGKSLEWIGYIYPYNG GTGYNQRFKSKATLTVDNSSSTAYMELRSLTSEDSAVYYCARLVYFDYWGLGTTLT VSS (SEQ ID NO:79), wherein the three CDRs (as determined by the IMGT numbering system) are in bolded italics.

[0197] In some embodiments, a CD3-binding molecule is provided, which includes a CD3-specific VL domain comprising three CDRs of the light chain CDRs of clone CD17.5F11, and a CD3-specific VH domain comprising three CDRs of the heavy chain CDRs of clone CD17.5F11. In some embodiments, a CD3-binding molecule may include a CD3-specific VL domain comprising three CDRs having an amino acid sequence of or set for in SEQ ID NO:102, YAS, and SEQ ID NO:104, respectively as well as a CD3-specific VH domain comprising three CDRs having an amino acid sequence of or set forth in SEQ ID NOs:99-101.

[0198] Additional embodiments provide a canine CD3-binding molecule, which includes: a CD3-specific VL domain and / or a CD3-specific VH domain of clone CD17.3G9 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CD17.3G9. In some embodiments, a canine CD3-binding molecule includes a CD3-specific VL domain and a CD3-specific VH domain of clone CD17.3G9.

[0199] Amino acid sequence of the VL domain of clone CD17.3G9: IIVMTQSPKSMSMSVGERVTLSCKASENVDSFVSWYQQKPEQSPKLLIFGASNRSTG VPDRFTGSGSTTDFTLTITSVQSEDLATYHCGQTYSFPFTFGSGTNLEIK (SEQ ID NO:74), wherein the three CDRs (as determined by the IMGT numbering system) are in bolded italics.

[0200] Amino acid sequence of the VH domain of clone CD17.3G9: EVQLVESGGGLVQPKGSLKLSCATSGFAFNIYAMNWVRQAPGKGLEWVARIRSKSK NYATYYAGSVKDRFIISRDDSQSMVYLHMNNLKTEDTAMYYCVRRGYFDVWGAGT TVTVSS (SEQ ID NO:73), wherein the three CDRs (as determined by the IMGT numbering system) are in bolded italics.4901-5416-2522.3Page 49 of 75 067505-000018WOPT

[0201] In some embodiments, a CD3-binding molecule is provided, which includes a CD3-specific VL domain comprising three CDRs of the light chain CDRs of clone CD17.3G9, and a CD3-specific VH domain comprising three CDRs of the heavy chain CDRs of clone CD17.3G9. In some embodiments, a CD3-binding molecule may include a CD3-specific VL domain comprising three CDRs having an amino acid sequence of or set forth in SEQ ID NO:84, GAS, and SEQ ID NO:86, respectively, as well as a CD3-specific VH domain comprising three CDRs having an amino acid sequence of or set forth in SEQ ID NOs:81-83, respectively.

[0202] Additional embodiments provide a canine CD3-binding molecule, which includes: a CD3-specific VL domain and / or a CD3-specific VH domain of clone CD17.6B3 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CD17.6B3. In some embodiments, a canine CD3-binding molecule includes a CD3-specific VL domain and a CD3-specific VH domain of clone CD17.6B3.

[0203] Amino acid sequence of the VL domain of clone CD17.6B3: NIVMTQSPKSMSMSVGERVTLNCKASENVDSYVSWYQQKPKQSPTLLIYGASNRST GVPDRFTGSGSVTDFTLTITSVQAEDLAYYHCGQTYSYPFTFGSGTKLEIK (SEQ ID NO:78), wherein the three CDRs (as determined by the IMGT numbering system) are in bolded italics.

[0204] Amino acid sequence of the VH domain of clone CD17.6B3: EVQLVESGGGLVQPKGSLTLSCAASGFTFNIFAMNWVRQAPGKGLEWVARIRSKN NYFATYYAASVRDRFTISRDDSQSMVYLQMNNLKTEDTGMYYCVRRGYFDVWGA GTTVTVSS (SEQ ID NO:77), wherein the three CDRs (as determined by the IMGT numbering system) are in bolded italics.

[0205] In some embodiments, a CD3-binding molecule is provided, which includes a CD3-specific VL domain comprising three CDRs of the light chain CDRs of clone CD17.6B3, and a CD3-specific VH domain comprising three CDRs of the heavy chain CDRs of clone CD17.6B3. In some embodiments, a CD3-binding molecule may include a CD3-specific VL domain comprising three CDRs having an amino acid sequence of or set forth in SEQ ID NO:96, GAS, and SEQ ID NO:98, respectively, as well as a CD3-specific VH domain comprising three CDRs having an amino acid sequence of or set forth in SEQ ID NOs:93-95, respectively.

[0206] Additional embodiments provide a canine CD3-binding molecule, which includes: a CD3-specific VL domain and / or a CD3-specific VH domain of clone CD17.9C5 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CD17.9C5. In4901-5416-2522.3Page 50 of 75 067505-000018WOPTsome embodiments, a canine CD3-binding molecule includes a CD3-specific VL domain and a CD3-specific VH domain of clone CD17.9C5.

[0207] Amino acid sequence of the VL domain of clone CD17.9C5: NIVMTQSPKSMSMSVGERVTLNCKASENVDSYVSWYQQKPKQSPTLLIYGASNRST GVPDRFTGSGSVTDFTLTITSVQAEDLAYYHCGQTYSYPFTFGSGTKLEIK (SEQ ID NO:76), wherein the three CDRs (as determined by the IMGT numbering system) are in bolded italics.

[0208] Amino acid sequence of the VH domain of clone CD17.9C5: EVQLVESGGGLVQPKGSLTLSCAASGFTFNIFAMNWVRQAPGKGLEWVARIRSKN NYFATYYAASVRDRFTISRDDSQSMVYLQMNNLKTEDTGMYYCVRRGYFDVWGA GTTVTVSS (SEQ ID NO:75), wherein the three CDRs (as determined by the IMGT numbering system) are in bolded italics.

[0209] In some embodiments, a CD3-binding molecule is provided, which includes a CD3-specific VL domain comprising three CDRs of the light chain CDRs of clone CD17.9C5, and a CD3-specific VH domain comprising three CDRs of the heavy chain CDRs of clone CD17.9C5. In some embodiments, a CD3-binding molecule may include a CD3-specific VL domain comprising three CDRs having an amino acid sequence of or set forth in SEQ ID NO:90, GAS, and SEQ ID NO:92, respectively, as well as a CD3-specific VH domain comprising three CDRs having an amino acid sequence of or set forth in SEQ ID NOs:87-89, respectively.

[0210] CA17.9C5 and CA17.6B3 are separate hybridomas (i.e., from plate 9 and plate 6). They are physically separated. They turn out to have a same coding DNA sequence, hence a same peptide sequence.

[0211] In some embodiments, a canine CD3-binding molecule includes a CD3-specific VL domain and a CD3-specific VH domain of clone CD17.6F9 or a variant having at least 80%, 85%, 90%, or 95% sequence identity thereto. In some embodiments, a variant of clone CD17.6F9 is one whose VL domain contains a I1D substitution according to the IMGT numbering system. Amino acid sequence of the VL domain of clone CD17.6F9 is shown in SEQ ID NO:10, wherein the three L-CDRs (as determined by the IMGT numbering system) havE an amino acid sequence of or set forth in SEQ ID NO:108, GAS, and SEQ ID NO:110, respectively. Amino acid sequence of the VH domain of clone CD17.6F9 is shown in SEQ ID NO:9, wherein the three CDRs (as determined by the IMGT numbering system) are shown in SEQ ID NOs:105-107, respectively.4901-5416-2522.3Page 51 of 75 067505-000018WOPT

[0212] In some embodiments, a canine CD3-binding molecule includes a CD3-specific VL domain and a CD3-specific VH domain of clone CD17.8D4 or a variant having at least 80%, 85%, 90%, or 95% sequence identity thereto. Amino acid sequence of the VL domain of clone CD17.8D4 is shown in SEQ ID NO:8, wherein the three L-CDRs (as determined by the IMGT numbering system) have an amino acid sequence of SEQ ID NO:161, GAS, and SEQ ID NO:163, respectively. Amino acid sequence of the VH domain of clone CD17.8D4 is shown in SEQ ID NO:7, wherein the three H-CDRs (as determined by the IMGT numbering system) have an amino acid sequence shown in SEQ ID NOs:158-160, respectively.

[0213] In some embodiments, a canine CD3-binding molecule includes a CD3-specific VL domain and a CD3-specific VH domain of clone CD17.2A12 or a variant having at least 80%, 85%, 90%, or 95% sequence identity thereto. Amino acid sequence of the VL domain of clone CD17.2A12 is shown in SEQ ID NO:6. Amino acid sequence of the VH domain of clone CD17.2A12 is shown in SEQ ID NO:5, wherein the three H-CDRs (as determined by the IMGT numbering system) have an amino acid sequence shown in SEQ ID NOs:164-166, respectively.

[0214]

[0215] In preferable embodiments, the canine CD3-binding molecule is a humanized antibody. In some embodiments, the canine CD3-binding molecule further comprises a constant domain (CL) of human Ig kappa light chain, and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG1 heavy chain.

[0216] In preferable embodiments, the canine CD3-binding molecule is a humanized antibody. In some embodiments, the canine CD3-binding molecule further comprises a constant domain (CL) of human Ig kappa light chain, and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG2 heavy chain.

[0217] In various embodiments, the immunogen of CD3-specific antibodies disclosed herein can be generated by immunizing mice with the T cell receptor complex (TCR / CD3) isolated from canine thymocytes; the antibody-producing B cells of the mice are fused with immortal myeloma cells to create hybridomas; and these hybrid cells are cloned to produce the desired antibody. In other embodiments, the CD3-binding molecule disclosed herein can be generated through recombinant DNA techniques, where genes for the antibody are inserted into host cells for large-scale production. Once generated, the antibody may be sequenced to identify or confirm sequence. For example, RNA can be extracted from the hybridoma cell pellets, and cDNA was created from the RNA by reverse-transcription with an oligo(dT) primer. PCR reactions are set up using variable domain primers to amplify both the VH and VL regions of the monoclonal antibody DNA. The VH and VL products can then be cloned4901-5416-2522.3Page 52 of 75 067505-000018WOPTinto the sequencing vector (e.g., Invitrogen pCR2.1) and transformed into TOP10 cells and screened by PCR for positive transformants. Selected colonies can then be picked and analyzed by DNA sequencing.

[0218] In traditional immune function, the interaction of antibody-antigen complexes with cells of the immune system is initiated through the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes results from the structural heterogeneity of the three Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A) and FcγRIII (CD16) are activating (i.e., immune system enhancing) receptors; FcγRIIB (CD32B) is an inhibiting (i.e., immune system dampening) receptor. The amino acid sequence of a human IgG1 Fc region is set forth in SEQ ID NO:25. The amino acid sequence of a mouse IgG1 Fc region is set forth in SEQ ID NO:21 or 24.

[0219] The present disclosure includes antibodies that specifically bind to CD3 that comprise a variant Fc region having Fc regions that are modified (e.g., substitutions, deletions, insertions in one or more portions) so as to be unable or less able to bind to the Fc receptor (relative to an antibody having the same CDRs but a wild-type Fc region). Fc receptor (FcR)- non-binding CD3-specific antibodies are believed to be minimally depleting. In some embodiments, a variant Fc region is provided which comprises at least one amino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH1 domain of the Fc region. In some embodiments, a variant Fc region is provided which comprises at least one amino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain of the Fc region. In some embodiments, a variant Fc region is provided which comprises at least one amino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH3 domain of the Fc region. The Fc variants of the present disclosure may be combined with other Fc modifications, such as those disclosed in U.S. Pat. Nos. 7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008; 7,335,742; 7,332,581; 7,183,387; 7,122,637; and 6,737,056; in PCT Publications Nos. WO 2008 / 105886; WO 2008 / 002933; WO 2007 / 021841; WO 2007 / 106707; WO 06 / 088494; WO 05 / 115452; WO 05 / 110474; WO 04 / 1032269; and in WO 04 / 063351. In certain embodiments, the antibody of the invention comprises a variant Fc region (including an Fc derived from any human immunoglobulin type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass), wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type4901-5416-2522.3Page 53 of 75 067505-000018WOPTFc region, which variant Fc region exhibits reduced or abolished binding to one or more effector ligands as determined by standard assays known in the art and disclosed herein, relative to a comparable molecule comprising the wild type Fc region.

[0220] In some embodiments, a canine CD3-binding molecule includes: a CD3- specific VL domain and a CD3-specific VH domain of one anti-dog CD3 antibody of clone CA17.6F9 or CA17.8D4 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CA17.6F9 or CA17.8D4, optionally the variant of the clone CD17.6F9 comprising an amino acid substitution in the VL domain at the N-terminus replacing Ile (I) with Asp (D); and a constant domain (CL) of human Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG1 heavy chain; wherein the canine CD3-binding molecule does not include the VL or the VH domains of anti-dog CD3 antibody clone CA17.2A12.

[0221] Various embodiments provide a canine CD3-binding molecule, which includes: the 3 CDRs of a CD3-specific VL domain and the 3 CDRs of a CD3-specific VH domain of one same anti-dog CD3 antibody selected from clone CD17.2A12, CA17.6F9, or CA17.8D4; and optionally further a constant domain (CL) of human Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG1 heavy chain.

[0222] In some embodiments, a canine CD3-binding molecule includes: the 3 CDRs of a CD3-specific VL domain and the 3 CDRs of a CD3-specific VH domain of one same anti-dog CD3 antibody of CA17.6F9 or CA17.8D4; and optionally further a constant domain (CL) of human Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG1 heavy chain; wherein the canine CD3-binding molecule does not include the VL and VH domains of anti-dog CD3 antibody clone CA17.2A12.

[0223] In other embodiments, a canine CD3-binding molecule contains the three constant domains and the hinge region of human IgG2, human IgG3, or human IgG4 heavy chain.

[0224] Some embodiments provide a canine CD3-binding molecule, which includes: a CD3-specific VL domain and a CD3-specific VH domain of one anti-dog CD3 antibody selected from clone CD17.2A12, CA17.6F9, or CA17.8D4 or a variant of the clone CD17.6F9 comprising an amino acid substitution in the VL domain at the N-terminus from Ile (I) to Asp (D); and4901-5416-2522.3Page 54 of 75 067505-000018WOPTa constant domain (CL) of a mouse Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of a mouse IgG1 or IgG2A heavy chain.

[0225] Some embodiments provide a canine CD3-binding molecule, which includes: the 3 CDRs of a CD3-specific VL domain and the 3 CDRs of a CD3-specific VH domain of one same anti-dog CD3 antibody selected from clone CD17.2A12, CA17.6F9, or CA17.8D4; and a constant domain (CL) of a mouse Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of a mouse IgG1 or IgG2A heavy chain.

[0226] In some embodiments, a canine CD3-binding molecule includes: a CD3- specific VL domain and a CD3-specific VH domain of one anti-dog CD3 antibody of clone CA17.5F11 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CA17.5F11; and a constant domain (CL) of human Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG1 heavy chain.

[0227] In some embodiments, a canine CD3-binding molecule includes: a CD3- specific VL domain and a CD3-specific VH domain of one anti-dog CD3 antibody of clone CA17.3G9 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CA17.3G9; and a constant domain (CL) of human Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG1 heavy chain.

[0228] In some embodiments, a canine CD3-binding molecule includes: a CD3- specific VL domain and a CD3-specific VH domain of one anti-dog CD3 antibody of clone CA17.6B3 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CA17.6B3; and a constant domain (CL) of human Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG1 heavy chain.

[0229] In some embodiments, a canine CD3-binding molecule includes: a CD3- specific VL domain and a CD3-specific VH domain of one anti-dog CD3 antibody of clone CA17.9C5 or a variant having at least 80%, 85%, 90%, or 95% sequence identity to the clone CA17.9C5; and a constant domain (CL) of human Ig kappa light chain and three constant domains (CH1, CH2, CH3) and a hinge region of human IgG1 heavy chain.

[0230] In some embodiments, the CL of the human Ig kappa light chain has an amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:26), or at least 95%, 90%, 85%, 80%, or 75% sequence identity thereto.4901-5416-2522.3Page 55 of 75 067505-000018WOPT

[0231] In some embodiments, the three constant domains and the hinge region of the human IgG1 heavy chain has an amino acid sequence (CH1-hinge-CH2-CH3) of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:25), or at least 95%, 90%, 85%, 80%, or 75% sequence identity thereto.

[0232] In various embodiments, the CD3-binding molecule is an antibody. In some embodiments, the CD3-binding molecule is a humanized antibody.

[0233] In various embodiments, the canine CD3-binding molecule is capable of immunospecifically binding to and activating canine T cells, characterized by expression of activation marker CD25 in the canine T cells. In various embodiments, the provided canine CD3 antibodies with the human IgG1 Fc domain activates T-cells higher than one with rat IgG Fc domain.

[0234] Some embodiments provide a vector encoding a canine CD3-binding molecule disclosed herein. In some embodiments, the vector further encodes a first secretory signal sequence at the N-terminus relative to the CD3-specific VL domain and / or a second secretory signal sequence at the N-terminus relative to the CD3-specific VH domain, optionally having an amino acid sequence shown in figure 1E, 1F or 1G.

[0235] A cell expression system is also provided, comprising a cell engineered with a vector disclosed herein; optionally the cell being a hybridoma.

[0236] Various embodiments provide methods of inducing canine T cell activation, which include treating canine T cells or canine peripheral blood mononuclear cells (PBMCs) with a canine CD3-binding molecule disclosed herein, wherein at least a portion of the canine PBMCs comprise T cells.

[0237] In some embodiments, the method of inducing canine T cell activation further includes measuring an increased expression of CD25 in the canine T cells or canine PBMCs in response to the treatment with the canine CD3-binding molecule.

[0238] In some embodiments, a multispecific antibody construct or a canine CD3- binding molecule contains an IgG1 type heavy chain modified to substitute MALAPG (SEQ ID NO:150) for LALAPG (SEQ ID NO:151) to inhibit interaction with Fc receptor of canine IgG1. In other embodiments, a multispecific antibody construct contains a Fc-silenced, IgG14901-5416-2522.3Page 56 of 75 067505-000018WOPTtype heavy chain. While the variable fragments (Fvs) of the heavy and light chains mediate specific target binding, antibodies possess effector functions through binding of the constant region (Fc) to Fcγ receptors (FcγR), which allow immune cells to attack and kill target cells using a variety of mechanisms. However, for some applications, including T-cell-engaging bispecifics, this effector function is typically undesired. Mutations within the lower hinge and the second constant domain (CH2) of IgG1 that comprise the FcγR binding interface reduce or eliminate effector function (“Fc silencing”) while retaining binding to the neonatal Fc receptor (FcRn), important for normal antibody pharmacokinetics (PKs). For example, some CH2 mutations, including D265A and P331S, are effective in reducing binding to FcγR but may reduce stability, promoting aggregation, particularly under acidic conditions. In some embodiments, the fragment crystallizable (Fc) region contains a mutation effective for eliminating Fc effector function with the retention of wild type-like stability, including L234A, L235A, and S267K (LALA-S267K), L234A, L235E, and S267K (LALE-S267K), L234A, L235A, and P329A (LALA-P329A), and L234A, L235E, and P329G (LALE-P329G). Further description of Fc-silencing or Fc-silenced IgG is provided by R. Pejchal et al., Antibodies (Basel), 2023 Aug 22;12(3):54.

[0239] In various embodiments, a CD3-binding molecule disclosed herein may be used in methods of treating, preventing, slowing the progression of and / or ameliorating the symptoms of T-cell mediated diseases or disorders, including graft rejection, graft versus host disease, unwanted delayed-type hypersensitivity reactions (such as delayed-type allergic reactions), T-cell mediated pulmonary diseases, and autoimmune diseases. T-cell mediated pulmonary diseases include sarcoidosis, hypersensitivity pneumonitis, acute interstitial pneumonitis, alveolitis, pulmonary fibrosis, idiopathic pulmonary fibrosis and other diseases characterized by inflammatory lung damage. T-cell autoimmune diseases include multiple sclerosis, neuritis, polymyositis, psoriasis, vitiligo, Sjogren's syndrome, rheumatoid arthritis, Type 1 diabetes, autoimmune pancreatitis, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), celiac disease, glomerulonephritis, scleroderma, sarcoidosis, autoimmune thyroid diseases (e.g., Hashimoto's thyroiditis and Graves' disease), myasthenia gravis, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, and systemic lupus erythematosus, lupus (particularly, cutaneous), effects from organ transplantation, graft vs. host disease (GVHD), etc. Particularly, the methods of the invention are advantageous in subjects with early stage disease to slow or reduce the damage from the autoimmunity and maintain a high level of function and / or reduce the need for other therapy (e.g., in the treatment or prophylaxis of Type I diabetes, the methods4901-5416-2522.3Page 57 of 75 067505-000018WOPTof the invention may reduce the need for exogenous insulin administration in the subject). In addition, the methods of the invention may advantageously reduce the incidence of or result in no incidence of cytokine release syndrome previously associated with administration of therapeutic antibodies, and, in particular, anti-T-cell (e.g., anti-CD3 antibody or antigen- binding fragments).

[0240] In certain embodiments, the course of treatment with an anti-CD3 antibody or CD3-binding molecule in the methods is repeated at 2-month, 4-month, 6-month, 8-month, 9- month, 10-month, 12-month, 15-month, 18-month, 24-month, 30-month, or 36-month intervals. In specific embodiments, efficacy of the treatment with an anti-CD3 antibody or antigen-binding fragments is determined at 2 months, 4 months, 6 months, 9 months, 12 months, 15 months, 18 months, 24 months, 30 months, or 36 months subsequent to the previous treatment.

[0241] In another embodiment, a subject is administered one or more unit doses of approximately 0.5-50 μg / kg, approximately 0.5-40 μg / kg, approximately 0.5-30 μg / kg, approximately 0.5-20 μg / kg, approximately 0.5-15 μg / kg, approximately 0.5-10 μg / kg, approximately 0.5-5 μg / kg, approximately 1-5 μg / kg, approximately 1-10 μg / kg, approximately 20-40 μg / kg, approximately 20-30 μg / kg, approximately 22-28 μg / kg or approximately 25-26 μg / kg of one or more anti-CD3 antibody or antigen-binding fragments to prevent, treat or ameliorate one or more symptoms of an autoimmune disorder or T cell malignancy. In another embodiment, a subject is administered one or more unit doses of 200 μg / kg, 178 μg / kg, 180 μg / kg, 128 μg / kg, 100 μg / kg, 95 μg / kg, 90 μg / kg, 85 μg / kg, 80 μg / kg, 75 μg / kg, 70 μg / kg, 65 μg / kg, 60 μg / kg, 55 μg / kg, 50 μg / kg, 45 μg / kg, 40 μg / kg, 35 μg / kg, 30 μg / kg, 26 μg / kg, 25 μg / kg, 20 μg / kg, 15 μg / kg, 13 μg / kg, 10 μg / kg, 6.5 μg / kg, 5 μg / kg, 3.2 μg / kg, 3 μg / kg, 2.5 μg / kg, 2 μg / kg, 1.6 μg / kg, 1.5 μg / kg, 1 μg / kg, 0.5 μg / kg, 0.25 μg / kg, 0.1 μg / kg, or 0.05 μg / kg of one or more anti-CD3 antibody or antigen-binding fragments to prevent, treat or ameliorate one or more symptoms of an autoimmune disorder or T-cell malignancy.

[0242] In additional embodiments, a CD3-binding molecule is used in the context of a multispecific antibody construct, such as a bispecific or trispecific T-cell engager, for a method of treating cancer as disclosed herein.

[0243] Yet in further embodiments, antibodies to CD3 made by the methods disclosed herein may also be used to identify the presence or absence of cancerous cells, or other diseased (non-cancerous) cells, or the level thereof, which are circulating in blood after their release from the cell surface (e.g., soluble CD3); or cancer stem cells in a variety of tissues. Such4901-5416-2522.3Page 58 of 75 067505-000018WOPTcirculating antigen may be an intact CD3 antigen, or a fragment thereof that retains the ability to be detected according to the methods taught herein. One method of using the antibodies for diagnosis is in vivo tumor imaging by linking the antibody to a radioactive or radio-opaque agent, administering the antibody to the individual and using an x-ray or other imaging machine to visualize the localization of the labeled antibody at the surface of cancer cells expressing the antigen. The antibody is administered at a concentration that promotes binding at physiological conditions. In vitro techniques for detection of CD3 include those routine in the art such as enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.

[0244] The compositions of the present invention may be administered by any suitable means, including parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and / or intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody may suitably be administered by pulse infusion, e.g., with increasing doses of the antibody. Preferably, the dosing is given intravenously or subcutaneously, and more preferably by intravenous infusion(s). Each exposure may be provided using the same or a different administration means. In one embodiment, each exposure is by intravenous administration. In another embodiment, each exposure is given by subcutaneous administration. In yet another embodiment, the exposures are given by both intravenous and subcutaneous administration.

[0245] Therapeutic formulations of the antibodies used in embodiments of the present invention are prepared for storage, shipment and administration by mixing a composition of the present invention having a desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers recognized in the pharmaceutical art in the form of lyophilized formulations or aqueous solutions.

[0246] The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch,4901-5416-2522.3Page 59 of 75 067505-000018WOPTglucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0247] The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of a prophylactic and / or therapeutic agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise a prophylactically or therapeutically effective amount of humanized antibodies of the invention and a pharmaceutically acceptable carrier.

[0248] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. Pharmaceutical compositions suitable for injection include sterile aqueous solutions where the active agents are water soluble, or dispersions or sterile powders for extemporaneous preparation of sterile injectable solutions. Compositions for use in the combination therapy may be prepared by incorporating the active antagonist or antibody in the required amount with appropriate carriers, for example water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride may be included in the composition.

[0249] The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those4901-5416-2522.3Page 60 of 75 067505-000018WOPTformed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0250] The invention provides a pharmaceutical pack or kit comprising one or more containers filled with humanized antibodies of the invention. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0251] The present disclosure also provides kits that can be used in the above methods. In one embodiment, a kit comprises one or more humanized antibodies of the invention. In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of cancer, in one or more containers. In another embodiment, a kit further comprises one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

[0252] In one embodiment, the present invention provides an article of manufacture containing antibodies to be used for the combined therapy for treatment of autoimmune disease. The article of manufacture comprises a container comprising a first antibody that binds an antigen present on a T cell and a pharmaceutically acceptable carrier, excipient or diluent within the container. The article of manufacture further comprises a second container comprising a second antibody directed to a B cell surface marker and a pharmaceutically acceptable carrier, excipient or diluent and instructions for administering the composition to a subject in need of treatment for autoimmune disease. Where the first and second antibodies are determined to be complementary and to not adversely affect each other, the first and the second antibody may be provided in a single container containing the first and second antibody in appropriate concentrations for administration together with a package insert and instructions for administration.

[0253] Containers of the article of manufacture may be of any suitable material that will not react with or otherwise affect the preparation. The article of manufacture may further comprise a second or a third container comprising a pharmaceutically-acceptable diluent4901-5416-2522.3Page 61 of 75 067505-000018WOPTbuffer, such as bacteriostatic water for injection, phosphate-buffered saline, Ringer's solution and dextrose solution. The article of manufacture may also include other material that may be desired from a commercial and user standpoint including other buffers, diluents, filters, needles and syringes. EXAMPLES

[0254] The following examples are provided to better illustrate the claimed disclosure and are not to be interpreted as limiting the scope of the disclosure. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the disclosure. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the disclosure. Example. Local Immunotherapy for pediatric brain tumors: preparing for POC clinical trial in canine brain tumor patients.

[0255] We conceive using Hydrogel-embedded Immunotherapy (HIT) to address the current challenges of treating pediatric brain tumor (the residual cancer cells, immunosuppressive environment, and on-target / off-cancer toxicity). HIT is cartilage-like protein-based hydrogel embedded with interferon-gamma (IFNg) and PDL1:CD3 Bi-specific T-cell engagers (BTE). We run canine clinical trials with companion canine cancer (oral melanoma, soft tissue sarcoma, and brain tumor) patients.

[0256] Brain tumors are the most common pediatric solid tumor and the leading cause of cancer-induced death in children. Current treatment including immunotherapy (IT) fail to achieve local control due to the remaining cancer cells, immunosuppressive environment, and on-target / off-cancer toxicity. Local IT can overcome these challenges if the cancer cells are exposed to therapeutics for a prolong period. Prolonged release from HIT’s hydrogel enables durable drug exposure after a single intra-operative administration.

[0257] Testing HIT requires an optimal animal model. Companion canine cancer patients have been recognized as reasonable subjects to test IT to translate the knowledge to humans. Their tumors are autochthonous, their immune systems are intact, and the patients represent the genetic heterogeneity observed in humans more closely than inbred mouse. Thus, we conceive to test HIT by using canine cancer patients.

[0258] Hydrogel is used as a drug delivery system because of its biocompatibility, biodegradability, and ability to control release. Additionally, our coiled-coiled cartilage-like recombinant protein hydrogel has superior properties for clinical use. a) Multiple protein-based immunotherapies can be easily loaded by reconstituting the lyophilized protein (the base of the hydrogel) with therapies-containing PBS. b) This peptide sequence-based coiled-coil4901-5416-2522.3Page 62 of 75 067505-000018WOPTinteraction offers tunability and injectability. c) This hydrogel has superior release kinetics profiles that can be easily tuned by changing weight percent of the hydrogel for optimal delivery of therapeutic molecules (Figure 9).

[0259] Bi-specific T-cell engagers (BTE) is a form of immunotherapy that is created by linking target regions for cancer cells (ex. PDL1, B7H3) and T-cells (ex. CD3). BTE binds both cancer cells and T-cells, redirecting T-cells to attack cancer cells. We designed 12 canine canonical BTE (ccBTE) and eight of them got produced. To show the efficacy of these BTEs on cancer cells, we set up a T-cell killing assay (TCK) with our BTE, canine glioblastoma (GBM), and activated T-cells (ATCs) and showed that all the eight BTEs were efficacious. We also showed that these BTEs were concentration-dependent. To show the combined effect of PDL1:CD3 BTE and IFNg, we set up TCK with PDL1:CD3 BTE and IFNg and showed that those two jointly killed canine GBM cells. As shown in figures 5C-5E, ccPD-L1:CD3 BTE + canine IFNg kills canine brain tumors. ccBTE has ScFv of a cancer binder and ScFv of CD3, connected by a linker. Figure 5C shows screening of 8 ccBTEs on canine GBM cells; 5D shows cBTE induces ATC-mediated cell death in GBM cells in a dose-dependent manner; and 5E shows ccBTE + canine IFNg jointly kills canine GBM cells. This cBTE + IFNg worked at least with GBM and melanoma cells.

[0260] To test our HIT, we are launching toxicity and clinical phase I safety studies in canine soft tissue sarcoma, brain tumor, melanoma (e.g., oral melanoma), and lymphoma. We conceive that these patients also receive surgical resections, following HIT, which will permit evaluation of pharmacodynamic responses and modification of therapeutic approaches (e.g., dose) as needed, (figure 10). To ensure canine companion patient’s health, vital signs (blood pressure & heart rate) will be monitored. To test the feasibility of HIT, IHC on CD25 (activated T-cell marker) and Granzyme (enzyme released from ATC) will be performed on resected tumor.

[0261] Overall, we engineered the HIT platform (cartilage-like protein-based hydrogel and canine PDL1:CD3 BTEs) and showed the combined effect of PDL1:CD3 BTE and IFNg.

[0262]

[0263] Various embodiments of the disclosure are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the4901-5416-2522.3Page 63 of 75 067505-000018WOPTspecification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0264] The foregoing description of various embodiments of the disclosure known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the disclosure to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the disclosure and its practical application and to enable others skilled in the art to utilize the disclosure in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the disclosure not be limited to the particular embodiments disclosed for carrying out the disclosure.

[0265] While particular embodiments of the present disclosure have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this disclosure and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this disclosure. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”4901-5416-2522.3Page 64 of 75 067505-000018WOPT

Claims

1. WHAT IS CLAIMED IS:

1. A multispecific antibody construct, comprising: one or more of a first polypeptide comprising a canine CD3-binding molecule, and one or more of a second polypeptide capable of binding an antigen associated with a disease, wherein the canine CD3-binding molecule comprises complementarity- determining regions (CDRs) of a variable light (VL) domain and / or a variable heavy (VH) domain of an anti-dog CD3 antibody of clone CA17.8D4, CA17.6F9, or CA17.2A12 or an antibody having at least 95% sequence identity to the clone CA17.8D4, CA17.6F9, or CA17.2A12, wherein, when the antigen associated with a disease comprises B7-H3, the canine CD3-binding molecule comprises the CDRs of the clone CA17.8D4, CA17.6F9, or both or does not comprise the CDRs of the clone CA17.2A12.

2. The multispecific antibody construct of claim 1, wherein the first polypeptide comprises a VHH, scFv, or Fab derived from the clone CA17.8D4.

3. The multispecific antibody construct of claim 1, wherein the second polypeptide comprises a VHH, scFv, or Fab derived from an antibody specific for an canine antigen comprising PD-L1, the B7-H3, EphA2, CEACAM5, ROR1, HER2 / neu, CD20, PSMA, IGF-1R, EGFR, A33, Ep-CAM, or a combination thereof.

4. The multispecific antibody construct of claim 1, wherein the one or more first polypeptides is linked to the one or more second polypeptides via a GlySer linker.

5. The multispecific antibody construct of claim 1, wherein the first polypeptide and the second polypeptide are connected via a knob-in-hole heterodimeric IgG1 Fc.

6. A method of killing canine cancer cells or cancer cells in a canine subject in need thereof, the method comprising: treating the canine cancer cells with, or administering to the canine subject, a multispecific antibody construct of any one of claims 1-5. optionally further comprising additionally treating the canine cancer cells with interferon-gamma, or further administering interferon-gamma to the canine subject.

7. The method of claim 6, wherein the cancer cells comprise glioblastoma cells, melanoma cells, or soft-tissue sarcoma cells.4901-5416-2522.3Page 65 of 75 067505-000018WOPT8. A canine CD3-binding molecule, comprising: a CD3-specific VL domain and / or a CD3-specific VH domain of one anti-dog CD3 antibody selected from clone CD17.2A12, CA17.6F9, CA17.8D4, or a variant of having at least 95% sequence identity to the clone CD17.2A12, CA17.6F9, CA17.8D4, wherein the variant comprises a variant of the CD17.6F9 which comprises an amino acid substitution at the N-terminus of its CD3-specific VL domain replacing Ile (I) with Asp (D); and a constant domain (CL) of a human Ig kappa light chain connected to the CD3- specific VL domain, and / or three constant domains (CH1, CH2, CH3) and a hinge region of a human IgG1 heavy chain connected to the CD3-specific VH domain.

9. The canine CD3-binding molecule of claim 8, wherein the CL of the human Ig kappa light chain has an amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO:26); and wherein the three constant domains and the hinge region of the human IgG1 heavy chain has an amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO:25).

10. The canine CD3-binding molecule of claim 8, wherein the CD3-binding molecule is an antibody.

11. The canine CD3-binding molecule of claim 8, which is capable of immunospecifically binding to and activating canine T cells, characterized by expression of activation marker CD25 in the canine T cells.

12. A vector encoding the canine CD3-binding molecule of any one of claims 8-11, wherein the vector further encodes a first secretory signal sequence at the N-terminus relative to the CD3-specific VL domain and / or a second secretory signal sequence at the N- terminus relative to the CD3-specific VH domain, optionally the encoded polypeptide comprising an amino acid sequence of one of SEQ ID NOs:38-44.4901-5416-2522.3Page 66 of 75 067505-000018WOPT13. A cell expression system, comprising a cell engineered with a vector of claim 12; optionally the cell being a hybridoma.

14. A method of inducing canine T cell activation, comprising: treating canine T cells or canine peripheral blood mononuclear cells (PBMCs) with a canine CD3-binding molecule of any one of claims 8-11, wherein at least a portion of the canine PBMCs comprise T cells.

15. The method of claim 14, further comprising measuring an increased expression of CD25 in the canine T cells or canine PBMCs in response to the treatment with the canine CD3-binding molecule.

16. A composition, for use in canine subjects, comprising: a. a recombinant protein, comprising a flexible domain and two or more self- association domains, wherein the self-association domain comprises canine cartilage oligomeric matrix protein fragment, wherein each of the two or more self-association domains are structured, the structure comprising a coil, wherein the flexible domain comprises a pseudorepeat of amino acids and has an amino acid sequence consisting of random repeats of alanine, glutamic acid, glycine, proline, serine, and threonine; and one or both of: b. a multispecific antibody construct comprising one or more of a first polypeptide capable of binding canine CD3 and one or more of a second polypeptide capable of binding an antigen; and c. a cytokine optionally comprising interferon; wherein the composition is formulated as a hydrogel composition comprising a hydrogel matrix formed from the recombinant protein, and wherein the multispecific antibody construct and / or the cytokine are associated with the hydrogel matrix.

17. A composition, for use in canine subjects, comprising: a multispecific antibody construct comprising one or more of a first polypeptide capable of binding canine CD3 and one or more of a second polypeptide capable of binding an antigen, a cytokine, optionally the cytokine comprising interferon, and a polymeric matrix,4901-5416-2522.3Page 67 of 75 067505-000018WOPTwherein the cytokine and the multispecific antibody construct are released from the polymeric matrix over a time span of 1-6 weeks or longer.

18. The composition of claim 16, wherein the canine cartilage oligomeric matrix protein fragment has an amino acid sequence of GPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA (SEQ ID NO:61) or GPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVME (SEQ ID NO:63).

19. The composition of claim 16, wherein the first polypeptide capable of binding canine CD3 is a VHH, scFv, or Fab derived from an anti-dog CD3 antibody of clone CA17.2A12, CA17.6F9, CA17.8D4, or a variant of having at least 95% sequence identity to the clone CD17.2A12, CA17.6F9, CA17.8D4.

20. The composition of claim 16, wherein the recombinant protein further comprises one or more of: a linker, optionally positioned between the flexible domain and the two flanking self-association domains, a marker sequence comprising an amino acid sequence of DYKDE (SEQ ID NO:2), and a Sortag sequence comprising an amino acid sequence of LPXTG, X being any amino acid (SEQ ID NO:136); and / or wherein the multispecific antibody construct further comprises a fragment crystallizable region (Fc).

21. The composition of any one of claims 16 and 18-20, wherein the multispecific antibody construct and / or the cytokine are independently covalently linked to the recombinant protein via a degradable linker.

22. The composition of any one of claims 16 and 18-20, wherein the cytokine and the multispecific antibody construct are associated with the hydrogel matrix formed from the recombinant protein in different ways, optionally selected from noncovalent mixture and covalent linkage via degradable linker of different degradation rate, such that the cytokine and the multispecific antibody construct are releasable from the hydrogel matrix at different rates.

23. The composition of any one of claims 16 and 18-20, wherein the cytokine and the multispecific antibody construct are released from the hydrogel matrix formed from the recombinant protein over a time span of 1-6 weeks or longer.

24. A method of killing cancer cells in a canine subject in need thereof, comprising: administering a composition of claim 23 to the canine subject in need thereof.4901-5416-2522.3Page 68 of 75 067505-000018WOPT25. The method of claim 24, wherein the cancer cells comprise glioblastoma cells, melanoma cells, or soft-tissue sarcoma cells.

26. A method of treating cancer or an overgrowth disease in a canine subject in need thereof, comprising: administering a composition of claim 23 to the canine subject in need thereof.

27. The method of claim 26, wherein the composition is injected to tumor parenchyma or tumor resection cavity or peritumoral tissue of the subject.

28. A system, comprising: a polymeric matrix in a lyophilized form,; one or more of a first polypeptide capable of binding canine CD3 and one or more of a second polypeptide capable of binding an antigen, preferably a cancer antigen, optionally the one or more first polypeptides and the one or more second polypeptides being associated as one molecule; a cytokine comprising interferon gamma and / or interferon alpha; a biocompatible medium; and one or more containers.

29. The system of claim 28, wherein the polymeric matrix comprises a recombinant protein comprising a flexible domain and two or more self-association domains, wherein the self-association domain comprises canine cartilage oligomeric matrix protein fragment, and wherein the flexible domain comprises a pseudorepeat of amino acids, and each of the one or more self-association domains are structured; and wherein the system further comprises a syringe or an injection device.

30. A recombinant protein, based on canine cartilage sequence, wherein the recombinant protein comprises: a flexible domain and two or more self-association domains, said flexible domain being flanked by the two or more self-association domains, wherein the self-association domain comprises canine cartilage oligomeric matrix protein fragment, and wherein each of the two or more self-association domains are structured, the structure comprising a coil; and wherein the flexible domain comprises a pseudorepeat of amino acids and has an amino acid sequence consisting of random repeats of alanine, glutamic acid, glycine, proline, serine, and threonine, wherein the flexible domain comprises a quantity of 36- 1,728 amino acids.4901-5416-2522.3Page 69 of 75 067505-000018WOPT31. The recombinant protein of claim 30, wherein the canine cartilage oligomeric matrix protein fragment has an amino acid sequence of GPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA (SEQ ID NO:61).

32. The recombinant protein of claim 30, wherein the canine cartilage oligomeric matrix protein fragment has an amino acid sequence of GPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVME (SEQ ID NO:63).

33. The recombinant protein of claim 30, further comprising one or more of: a linker, optionally positioned between the flexible domain and the two flanking self-association domains, a cell adhesion motif, and a marker sequence comprising an amino acid sequence of DYKDE (SEQ ID NO:2).

34. A vector encoding the recombinant protein of any one of claims 30-33, wherein the vector further encodes a secretory signal sequence at the N-terminus relative to the recombinant protein, and optionally further encodes a polyhistidine sequence.

35. A mammalian cell expression system, comprising a mammalian cell engineered with a vector according to claim 34.

36. A CD3-binding molecule comprising an antigen-binding fragment of an antibody, wherein the antigen-binding fragment comprises a CD3-specific variable light (VL) domain and / or a CD3-specific variable heavy (VH) domain, wherein: the CD3-specific VL domain comprises three complementarity determining regions (CDRs) having an amino acid sequence of SEQ ID NO:102, YAS, and SEQ ID NO:104, respectively, and the CD3-specific VH domain comprises three CDRs having an amino acid sequence of SEQ ID NOs:99, 100, and 101, respectively, or the CD3-specific VL domain and the CD3-specific VH domain comprise the CDRs of clone CA17.5F11; the CD3-specific VL domain comprises three CDRs having an amino acid sequence of SEQ ID NO:84, GAS, and SEQ ID NO:86, respectively, and the CD3-specific VH domain comprises three CDRs having an amino acid sequence of SEQ ID NOs:81-83, respectively, or the CD3-specific VL domain and the CD3-specific VH domain comprise the CDRs of clone CA17.3G9; or the CD3-specific VL domain comprises three CDRs having an amino acid sequence of SEQ ID NO:90, GAS, and SEQ ID NO:92, respectively, and the CD3-specific VH domain comprises three CDRs having an amino acid sequence of SEQ ID NOs:87, 88, and 89,4901-5416-2522.3Page 70 of 75 067505-000018WOPTrespectively, or the CD3-specific VL domain and the CD3-specific VH domain comprise the CDRs of clone CA17.9C5 or clone CA17.6B3.

37. The CD3-binding molecule of claim 36, wherein: the CD3-specific VL domain comprises the amino acid sequence of SEQ ID NO:80, and the CD3-specific VH domain comprises the amino acid sequence of SEQ ID NO:79; the CD3-specific VL domain comprises the amino acid sequence of SEQ ID NO:74, and the CD3-specific VH domain comprises the amino acid sequence of SEQ ID NO:73; or the CD3-specific VL domain comprises the amino acid sequence of SEQ ID NO:76, and the CD3-specific VH domain comprises the amino acid sequence of SEQ ID NO:75; respectively.

38. The CD3-binding molecule of claim 36, which is humanized.

39. The CD3-binding molecule of claim 36, which comprises a human Ig kappa light chain polypeptide which is encoded by a nucleic acid encoding an amino acid sequence of SEQ ID NO:72; 66; 68; or 70; respectively.

40. The CD3-binding molecule of claim 36, which comprises a human IgG1 type heavy chain polypeptide which is encoded by a nucleic acid encoding an amino acid sequence of SEQ ID NO:71; 65; 67; or 69; respectively.

41. A vector encoding the CD3-binding molecule of claim 37, wherein the vector further encodes a first secretory signal sequence at the N-terminus relative to the CD3-specific VL domain and / or a second secretory signal sequence at the N-terminus relative to the CD3- specific VH domain, optionally the encoded polypeptide comprising an amino acid sequence of one of SEQ ID NOs:65-72.

42. A cell expression system, comprising a cell engineered with a vector of claim 41; optionally the cell being a hybridoma.

43. A method of inducing canine T cell activation, comprising: treating canine T cells or canine peripheral blood mononuclear cells (PBMCs) with a CD3-binding molecule of any one of claims 36-40, wherein at least a portion of the canine PBMCs comprise T cells.

44. The method of claim 43, further comprising measuring an increased expression of CD25 in the canine T cells or canine PBMCs in response to the treatment with the canine CD3- binding molecule.

45. A multispecific antibody construct, comprising: a first polypeptide comprising the CD3-binding molecule of claim 36 and4901-5416-2522.3Page 71 of 75 067505-000018WOPTa second polypeptide capable of immunospecifically binding an antigen associated with a disease.

46. The multispecific antibody construct of claim 45, wherein the first polypeptide is a VHH or scFv, or the first polypeptide comprises a Fab.

47. The multispecific antibody construct of claim 45, wherein the second polypeptide is a VHH or scFv or comprises a Fab, wherein the VHH, the scFv, or the Fab is derived from an antibody specific for a canine antigen.

48. The multispecific antibody construct of claim 45, wherein the second polypeptide is capable of immunospecifically binding to a tumor antigen expressed on a tumor cell, wherein the tumor cell is from a cancer selected from the group consisting of: breast cancer, prostate cancer, gastric cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma, and leukemia.

49. The multispecific antibody construct of claim 45, wherein the second polypeptide is capable of immunospecifically binding to a cell surface antigen, receptor or receptor ligand, wherein the cell surface antigen, receptor or receptor ligand comprises PD-L1, B7-H3, EphA2, CEACAM5, ROR1, HER2 / neu, CD20, PSMA, IGF-1R, EGFR, A33, Ep-CAM, or a combination thereof.

50. The multispecific antibody construct of claim 45, wherein the second polypeptide is capable of immunospecifically binding to a cell surface antigen, receptor or receptor ligand, wherein the cell surface antigen, receptor or receptor ligand is a molecule involved in a T cell B cell association, wherein the molecule involved in said T cell B cell association is selected from the group consisting of CD28, CD40, CD19, CD20, CD22, CD23, CD27, CD32B, CD38, CD79a, CD79b, CD80, CD86, LFA-I, LFA-3, and CFA-I, and a combination thereof.

51. The multispecific antibody construct of claim 45, wherein the first polypeptide is linked to the second polypeptide via a linker, optionally the linker being a GlySer linker.

52. The multispecific antibody construct of claim 51, wherein the first polypeptide comprises the CD3-binding molecule in a form of single-chain Fv (scFv), and wherein the second polypeptide is in the form of scFv.

53. The multispecific antibody construct of claim 45, wherein the first polypeptide and the second polypeptide are connected via a knob-in-hole heterodimeric IgG1 Fc, wherein the first polypeptide comprises the CD3-specific VL domain, the CD3-specific VH domain, and a first4901-5416-2522.3Page 72 of 75 067505-000018WOPTof a IgG1 type heavy chain, and wherein the second polypeptide comprises a VL domain specific for a cancer antigen, a VH domain specific for the cancer antigen, and a second of the IgG1 type heavy chain.

54. The multispecific antibody construct of claim 45, wherein the first polypeptide and / or the second polypeptide comprises a Fc-silenced, canine IgG1 type heavy chain.

55. A method of killing canine cancer cells or cancer cells in a canine subject in need thereof, the method comprising: treating the canine cancer cells with, or administering to the canine subject, a multispecific antibody construct of any one of claims 45-53; optionally further comprising treating the canine cancer cells with interferon-gamma, or further administering interferon-gamma to the canine subject.

56. The method of claim 55, wherein the cancer cells comprise glioblastoma cells, melanoma cells, or soft-tissue sarcoma cells.

57. A composition, for use in canine subjects, comprising: a. a recombinant protein, comprising a flexible domain and two or more self- association domains, wherein the self-association domain comprises canine cartilage oligomeric matrix protein fragment, wherein each of the two or more self-association domains are structured, the structure comprising a coil, wherein the flexible domain comprises a pseudorepeat of amino acids and has an amino acid sequence consisting of random repeats of alanine, glutamic acid, glycine, proline, serine, and threonine; and b. a multispecific antibody construct of any one of claims 45-53; and optionally further comprising: c. a cytokine, optionally the cytokine comprising interferon. wherein the composition is formulated as a hydrogel composition, wherein the hydrogel composition comprises a hydrogel matrix formed from the recombinant protein, and wherein the multispecific antibody construct and the cytokine are associated with the hydrogel matrix.

58. A composition, for use in canine subjects, comprising: a multispecific antibody construct of any one of claims 45-53; a cytokine, optionally the cytokine comprising interferon; and a polymeric matrix,4901-5416-2522.3Page 73 of 75 067505-000018WOPTwherein the cytokine and the multispecific antibody construct are released from the polymeric matrix over a time span of 1-6 weeks or longer.

59. The composition of claim 57, wherein the recombinant protein further comprises one or more of: a linker, optionally positioned between the flexible domain and the two flanking self-association domains, a marker sequence comprising an amino acid sequence of DYKDE (SEQ ID NO:2), and a Sortag sequence comprising an amino acid sequence of LPXTG (SEQ ID NO:136), X being any amino acid; and / or wherein the multispecific antibody construct further comprises a fragment crystallizable region (Fc).

60. The composition of claim 57, wherein the cytokine and the multispecific antibody construct are associated with the hydrogel matrix formed from the recombinant protein in different ways, optionally selected from noncovalent mixture and covalent linkage via degradable linker of different degradation rate, such that the cytokine and the multispecific antibody construct are releasable from the hydrogel matrix at different rates.

61. The composition of claim 57, wherein the cytokine and the multispecific antibody construct are released from the hydrogel matrix formed from the recombinant protein over a time span of 1-6 weeks or longer.

62. A method of killing cancer cells in a canine subject in need thereof, comprising: administering a composition of claim 61 to the canine subject in need thereof.

63. The method of claim 62, wherein the cancer cells comprise glioblastoma cells, melanoma cells, or soft-tissue sarcoma cells.

64. A method of treating cancer or an overgrowth disease in a canine subject in need thereof, comprising: administering a composition of claim 61 to the canine subject.

65. The method of claim 64, wherein the composition is injected to tumor parenchyma of the subject.

66. A system, comprising: a polymeric matrix in a lyophilized form; a multispecific antibody construct of claim 45; a cytokine comprising interferon gamma and / or interferon alpha; a biocompatible medium; and one or more containers.

67. The system of claim 66, further comprising a syringe or an injection device.4901-5416-2522.3Page 74 of 75 067505-000018WOPT

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